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Results for “DNA Replication Stress”

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At least 127 records · Page 7Linked to original sources

PubMed2000

Regulation of DNA replication during the cell cycle: roles of Cdc7 kinase and coupling of replication, recombination, and repair in response to replication fork arrest.

DNA replication is central to cell growth, development, and generation of tissues and organs. Recent advances in understanding replication machinery have revealed striking conservation of components involved in the processes of DNA replication, from yeasts to human. The conservation extends even to bacteria for some basic components of replication apparatus. Eukaryotic DNA replication is regulated at various stages to ensure strict regulation during cell cycle. We have identified a novel mammalian kinase, Cdc7-ASK (Activator of S phase Kinase), that plays a key role at the entry into S phase as a molecular switch for DNA replication. This kinase is specifically activated during S phase and triggers the firing of DNA replication by phosphorylating an essential DNA helicase component of the replication complex. Environmental stresses such as DNA damages or depletion of essential nutrients for DNA synthesis lead to unscheduled arrest of DNA replication forks. In bacteria, this leads to induction of altered modes of DNA replication, which may repair DNA damages, facilitate reassembly of replication machinery at the stalled replication fork, or do both. In eukaryotes, blocking replication forks usually induces both checkpoint responses, which prevent premature progression of cell cycle events before precise completion of the preceding cell cycle stage, and the recombinational repair system for the lesions. Possible common bases in recognition of stalled replication forks in bacteria and eukaryotes will be discussed. Furthermore, we will discuss the potential of replication and checkpoint proteins as targets of anticancer agents as well as possible novel technology for stem cell amplification through manipulation of DNA replication.

Animals↗

PubMed2005

Protein phosphatase 5 is required for ATR-mediated checkpoint activation.

In response to DNA damage or replication stress, the protein kinase ATR is activated and subsequently transduces genotoxic signals to cell cycle control and DNA repair machinery through phosphorylation of a number of downstream substrates. Very little is known about the molecular mechanism by which ATR is activated in response to genotoxic insults. In this report, we demonstrate that protein phosphatase 5 (PP5) is required for the ATR-mediated checkpoint activation. PP5 forms a complex with ATR in a genotoxic stress-inducible manner. Interference with the expression or the activity of PP5 leads to impairment of the ATR-mediated phosphorylation of hRad17 and Chk1 after UV or hydroxyurea treatment. Similar results are obtained in ATM-deficient cells, suggesting that the observed defect in checkpoint signaling is the consequence of impaired functional interaction between ATR and PP5. In cells exposed to UV irradiation, PP5 is required to elicit an appropriate S-phase checkpoint response. In addition, loss of PP5 leads to premature mitosis after hydroxyurea treatment. Interestingly, reduced PP5 activity exerts differential effects on the formation of intranuclear foci by ATR and replication protein A, implicating a functional role for PP5 in a specific stage of the checkpoint signaling pathway. Taken together, our results suggest that PP5 plays a critical role in the ATR-mediated checkpoint activation.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2024

FANCJ DNA helicase is recruited to the replisome by AND-1 to ensure genome stability.

FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition.

Humans↗

PubMed1996

Two distinct modes of strand unlinking during theta-type DNA replication.

DNA gyrase and topoisomerase IV (Topo IV) are type II bacterial DNA topoisomerases that show a high degree of similarity to each other yet appear to have different functions during DNA replication. We show here that the torsional stress generated during theta-type DNA replication in vitro can take the form of either positive supercoils ahead of the replication fork or catenane-like right-handed windings (precatenanes) of the two partially replicated duplexes behind the fork. Gyrase prefers to act on the former, whereas Topo IV prefers the latter. Removal of either form of positive winding can support nascent chain elongation, but only precatenane removal can support the final stages of DNA replication: processing of the late intermediate and daughter chromosome decatenation.

DNA Replication↗

PubMed2026

FANCM is required for the PAX3::FOXO1-driven oncogenic program in rhabdomyosarcoma.

Many cancers are driven by mutationally altered transcription factors (TFs) that rewire cells to an oncogenic state. Cells must activate specific mechanisms to tolerate the burden of oncogenic TF activity. To define such mechanisms, we focused on a canonical oncogenic fusion protein-driven cancer, alveolar rhabdomyosarcoma (ARMS), where the PAX3::FOXO1 fusion protein hyperactivates and mislocalizes PAX3 and FOXO1 TF functions. Employing sequential functional genomic CRISPR-Cas9 screens, we identified FANCM, a DNA translocase in the Fanconi anemia pathway, as a selective dependency in PAX3::FOXO1+ ARMS. FANCM loss reduces fusion protein levels, induces myogenic differentiation, and disrupts the PAX3::FOXO1 transcriptional program, thereby halting oncogenic proliferation. Mechanistically, FANCM depletion exacerbates replication stress (RS) and DNA damage signaling, with chromatin-associated RS enriched at PAX3::FOXO1 target gene loci, resulting in selective downregulation of the oncogenic program. CRISPR exon-tiling screens prioritized FANCM's helicase and DNA-binding domains as essential for this dependency, linking FANCM-mediated replication fork binding to sustained oncogenesis.

ARMS↗

PubMed1988

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions.

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).

Animals↗

PubMed2005

Gemcitabine-induced activation of checkpoint signaling pathways that affect tumor cell survival.

Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

Diminished S-phase cyclin-dependent kinase function elicits vital Rad53-dependent checkpoint responses in Saccharomyces cerevisiae.

Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Delta cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Delta cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Delta cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Delta cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.

CDC2 Protein Kinase↗

PubMed2003

Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways.

The fidelity of DNA replication and repair processes is critical for maintenance of genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production and thus plays an essential role in DNA synthesis. The level and activity of RNR are highly regulated by the cell cycle and DNA damage checkpoints, which maintain optimal dNTP pools required for genetic fidelity. RNRs are composed of a large subunit that binds the nucleoside diphosphate substrates and allosteric effectors and a small subunit that houses the di-iron tyrosyl radical cofactor essential for the reduction process. In Saccharomyces cerevisiae, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). Here we report the subcellular localization of Rnr1-4 during normal cell growth and the redistribution of Rnr2 and Rnr4 in response to DNA damage and replicational stress. During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm and Rnr2 and Rnr4 are predominantly present in the nucleus. Under genotoxic stress, Rnr2 and Rnr4 become redistributed to the cytoplasm in a checkpoint-dependent manner. Subcellular redistribution of Rnr2 and Rnr4 can occur in the absence of the transcriptional induction of the RNR genes after DNA damage and likely represents a posttranslational event. These results suggest a mechanism by which DNA damage checkpoint modulates RNR activity through the temporal and spatial regulation of its subunits.

DNA Damage↗

PubMed2006

Circadian tempo: a paradigm for genome stability?

Circadian clocks are molecular time-keeping systems that underlie daily biological rhythms in anticipation of the changing light and dark cycles. These clocks mediate daily rhythms in physiology and behavior that are thought to confer an adaptive advantage for organisms. It is hypothesized that cell cycle checkpoints are gated to an intrinsic circadian clock to protect DNA from diurnal exposure to mutagens (e.g.; UV radiation peaks with daylight and dissolved genotoxins that fluctuate with feeding periods). It is proposed that DNA replication arrest in response to genotoxic stress is a likely basis for the evolution of circadian-gated DNA replication. This protective mechanism is highly conserved and can be traced along the evolutionary time-line to the early prokaryotes, unicellular eukaryotes and viruses. Peak DNA repair capacity is normally synchronous to the crest of mutagenic stress as they oscillate with respect to time. Mutator phenotypes with increased vulnerability to genotoxic stress may therefore develop when the circadian pattern of cell cycle control, DNA repair or apoptotic response are phase-shifted relative to the rhythm of mutagenic stress. The accumulating mutations would lead to accelerated aging, genome instability and neoplasia. The proposed model delineates areas of research with potentially profound implications for carcinogenesis.

Aging↗

PubMed2005

Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models.

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.

ATP-Binding Cassette Transporters↗

PubMed2001

The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress.

The lung is a major target tissue for oxidative stress, including hyperoxia used to relieve tissue hypoxia. Unfortunately, severe hyperoxia damages DNA, inhibits proliferation, and kills cells, resulting in morbidity and mortality. Although hyperoxia induces the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), their role in pulmonary injury remains unknown. Using p53- and p21-deficient mice we demonstrate that hyperoxia induces p21 in the absence of p53, suggesting that previous conclusions that p53 does not modify hyperoxic lung injury cannot be extrapolated to p21. In fact, mean survival of p21-deficient mice decreased by 40% and was associated with terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining of alveolar debris, indicative of DNA fragmentation and cell death. Ultrastructural analyses revealed that alveolar endothelial and type I epithelial cells died rapidly by necrosis. Although hyperoxia decreased DNA replication in p21-wild-type lungs, it had no effect on replication in p21-deficient lungs. Our findings suggest that p21 protects the lung from oxidative stress, in part, by inhibiting DNA replication and thereby allowing additional time to repair damaged DNA. Our findings have implications for patients suffering from the toxic effects of supplemental oxygen therapies.

Animals↗

PubMed2006

DNA-damage control: Claspin destruction turns off the checkpoint.

The ATR-Claspin-Chk1 pathway is critical for turning on the cellular response to DNA damage and replication stress. Five recent reports uncover new mechanisms controlling the recovery phase of the checkpoint response, and introduce crucial roles for Claspin, Rad17 phosphorylation and the ubiquitin proteasome pathway in Chk1 signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Human Asf1 regulates the flow of S phase histones during replicational stress.

Maintenance of chromosomal integrity requires tight coordination of histone biosynthesis with DNA replication. Here, we show that extracts from human cells exposed to replication stress display an increased capacity to support replication-coupled chromatin assembly. While in unperturbed S phase, hAsf1 existed in equilibrium between an active form and an inactive histone-free pool, replication stress mobilized the majority of hAsf1 into an active multichaperone complex together with histones. This active multichaperone complex was limiting for chromatin assembly in S phase extracts, and hAsf1 was required for the enhanced assembly activity in cells exposed to replication stress. Consistently, siRNA-mediated knockdown of hAsf1 impaired the kinetics of S phase progression. Together, these data suggest that hAsf1 provides the cells with a buffering system for histone excess generated in response to stalled replication and explains how mammalian cells maintain a critical "active" histone pool available for deposition during recovery from replication stresses.

Animals↗

PubMed2005

Drug-induced mitochondrial toxicity.

Mitochondria play a critical role in generating most of the cell's energy as ATP. They are also involved in other metabolic processes such as urea generation, haem synthesis and fatty acid beta-oxidation. Disruption of mitochondrial function by drugs can result in cell death by necrosis or can signal cell death by apoptosis (e.g., following cytochrome c release). Drugs that injure mitochondria usually do so by inhibiting respiratory complexes of the electron chain; inhibiting or uncoupling oxidative phosphorylation; inducing mitochondrial oxidative stress; or inhibiting DNA replication, transcription or translation. It is important to test for mitochondrial toxicity early in drug development as impairment of mitochondrial function can induce various pathological conditions that are life threatening or can increase the progression of existing mitochondrial diseases.

Animals↗

PubMed2025

A transient mutational burst occurs during yeast colony development.

Characterizing the contribution of mutators to mutation accumulation is essential for understanding cellular adaptation and diseases like cancer. By measuring single and double mutation rates, including point mutations, segmental duplications, and reciprocal translocations, we found that wild-type yeast colonies exhibit double mutation rates up to 17 times higher than expected from experimentally determined single mutation rates. These double mutants retained wild-type mutation rates, indicating they originated from genetically normal cells that transiently expressed a mutator phenotype. Numerical simulations suggest that transient mutator subpopulations likely consist of less than a few thousand cells, and experience high-intensity mutational bursts for less than five generations. Most double mutations accumulated sequentially across cell cycles, with simultaneous acquisition being rare and likely linked to systemic genomic instability. Additionally, we explored the genetic control of transient hypermutation and found that the excess of double mutants can be modulated by replication stress and the DNA damage tolerance pathway. Our findings suggest that transient mutators play a significant role in genomic instability and contribute to the mutational load accumulating in growing isogenic populations.

Saccharomyces cerevisiae↗

PubMed2003

Human claspin is required for replication checkpoint control.

Claspin is a newly identified protein that regulates Chk1 activation in Xenopus. In the present study we investigated the role of human Claspin in the DNA damage/replication checkpoint in mammalian cells. We observed that human Claspin is a cell cycle regulated protein that peaks at S/G2 phase. Claspin localizes in the nuclei, but it only associates with Chk1 following replication stress or other types of DNA damage. In addition, Claspin is phosphorylated in response to replication stress, and this phosphorylation appears to be required for its association with Chk1. Moreover, Claspin interacts with the checkpoint proteins ATR and Rad9. Given that both the ATR and Rad9-Rad1-Hus1 complexes are involved in Chk1 activation, it is possible that Claspin works as an adaptor molecule bringing these molecules together. Using small interfering RNA technology, we have shown that down-regulation of Claspin expression inhibits Chk1 activation in response to replication stress. More importantly, down-regulation of Claspin augments the premature chromatin condensation induced by hydroxyurea, inhibits the UV-induced reduction of DNA synthesis, and decreases cell survival. Taken together, these data imply a potentially critical role for Claspin in replication checkpoint control in mammalian cells.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The p53 pathway: positive and negative feedback loops.

The p53 pathway responds to stresses that can disrupt the fidelity of DNA replication and cell division. A stress signal is transmitted to the p53 protein by post-translational modifications. This results in the activation of the p53 protein as a transcription factor that initiates a program of cell cycle arrest, cellular senescence or apoptosis. The transcriptional network of p53-responsive genes produces proteins that interact with a large number of other signal transduction pathways in the cell and a number of positive and negative autoregulatory feedback loops act upon the p53 response. There are at least seven negative and three positive feedback loops described here, and of these, six act through the MDM-2 protein to regulate p53 activity. The p53 circuit communicates with the Wnt-beta-catenin, IGF-1-AKT, Rb-E2F, p38 MAP kinase, cyclin-cdk, p14/19 ARF pathways and the cyclin G-PP2A, and p73 gene products. There are at least three different ubiquitin ligases that can regulate p53 in an autoregulatory manner: MDM-2, Cop-1 and Pirh-2. The meaning of this redundancy and the relative activity of each of these feedback loops in different cell types or stages of development remains to be elucidated. The interconnections between signal transduction pathways will play a central role in our understanding of cancer.

Amino Acid Sequence↗