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PubMed2001

The UV-inducible RNA-binding protein A18 (A18 hnRNP) plays a protective role in the genotoxic stress response.

We have previously shown that specific RNA-binding proteins (RBP) are activated by genotoxic stress. The role and function of these stress-activated RBP are, however, poorly understood. The data presented here indicate that the RBP A18 heterogeneous ribonucleoprotein (hnRNP) is induced and translocated from the nuclei to the cytoplasm after exposure to UV radiation. Using a new in vitro system we identified potential cellular targets for A18 hnRNP. Forty-six mRNA transcripts were identified, most of which are stress- or UV-responsive genes. Two important stress-responsive transcripts, the replication protein A (RPA2) and thioredoxin, were studied in more detail. Northwestern analyses indicate that A18 hnRNP binds specifically to the 3'-untranslated region of RPA2 transcript independently of its poly(A) tail, whereas the poly(A) tail of thioredoxin mRNA reinforces binding. Overexpression of A18 hnRNP increases the mRNAs stability and consequently enhances translation in a dose-dependent manner. Moreover, cell lines expressing reduced levels of A18 hnRNP are more sensitive to UV radiation. These data suggest that A18 hnRNP plays a protective role against genotoxic stresses by translocating to the cytosol and stabilizing specific transcripts involved in cell survival.

3' Untranslated Regions↗
PubMed2026

Metabolic regulation of mitochondrial DNA (mtDNA) homeostasis.

Mitochondria are central hubs of cellular metabolism that harbor their own genome (mtDNA), whose maintenance is essential for both cellular and organismal homeostasis. Unlike nuclear DNA, mtDNA replicates continuously throughout the cell cycle, rendering it particularly sensitive to changes in metabolic state. Emerging evidence indicates that mtDNA homeostasis is not governed solely by dedicated replication factors but is tightly coupled to cellular metabolism. In this review, we discuss how metabolic networks shape mtDNA maintenance through three interconnected layers: mitochondrial nucleotide pools, metabolic control of the replication machinery, and stress-response pathways. This conceptual framework underscores the direct role of metabolic state in governing mtDNA replication, stability, and quality control, with significant implications for mitochondrial disease and therapeutic strategies.

Integrated stress response (ISR)↗
PubMed2004

Disappearance of the telomere dysfunction-induced stress response in fully senescent cells.

Replicative senescence is a natural barrier to cellular proliferation that is triggered by telomere erosion and dysfunction. Here, we demonstrate that ATM activation and H2AX-gamma nuclear focus formation are sensitive markers of telomere dysfunction in primary human fibroblasts. Whereas the activated form of ATM and H2AX-gamma foci were rarely observed in early-passage cells, they were readily detected in late-passage cells. The ectopic expression of telomerase in late-passage cells abrogated ATM activation and H2AX-gamma focus formation, suggesting that these stress responses were the consequence of telomere dysfunction. ATM activation was induced in quiescent fibroblasts by inhibition of TRF2 binding to telomeres, indicating that telomere uncapping is sufficient to initiate the telomere signaling response; breakage of chromosomes with telomeric associations is not required for this activation. Although ATM activation and H2AX-gamma foci were readily observed in late-passage cells, they disappeared once cells became fully senescent, indicating that constitutive signaling from dysfunctional telomeres is not required for the maintenance of senescence.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length.

In Saccharomyces cerevisiae, DNA replication stress activates the replication checkpoint, which slows S-phase progression, stabilizes slowed or stalled replication forks, and relieves inhibition of the ribonucleotide reductase (RNR) complex. To identify novel genes that promote cellular viability after replication stress, the S. cerevisiae non-essential haploid gene deletion set (4812 strains) was screened for sensitivity to the RNR inhibitor hydroxyurea (HU). Strains bearing deletions in either CCR4 or CAF1/POP2, which encode components of the cytoplasmic mRNA deadenylase complex, were particularly sensitive to HU. We found that Ccr4 cooperated with the Dun1 branch of the replication checkpoint, such that ccr4Delta dun1Delta strains exhibited irreversible hypersensitivity to HU and persistent activation of Rad53. Moreover, because ccr4Delta and chk1Delta exhibited epistasis in several genetic contexts, we infer that Ccr4 and Chk1 act in the same pathway to overcome replication stress. A counterscreen for suppressors of ccr4Delta HU sensitivity uncovered mutations in CRT1, which encodes the transcriptional repressor of the DNA-damage-induced gene regulon. Whereas Dun1 is known to inhibit Crt1 repressor activity, we found that Ccr4 regulates CRT1 mRNA poly(A) tail length and may subtly influence Crt1 protein abundance. Simultaneous overexpression of RNR2, RNR3 and RNR4 partially rescued the HU hypersensitivity of a ccr4Delta dun1Delta strain, consistent with the notion that the RNR genes are key targets of Crt1. These results implicate the coordinated regulation of Crt1 via Ccr4 and Dun1 as a crucial nodal point in the response to DNA replication stress.

Cell Cycle Proteins↗
PubMed2005

DNA damage and replication stress induced transcription of RNR genes is dependent on the Ccr4-Not complex.

Genetic experiments have indicated a role for the Ccr4-Not complex in the response to hydroxyurea (HU) induced replication stress and ionizing radiation in yeast. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4 and degradation of its inhibitor, Sml1p. The Ccr4-Not complex has originally been described as a negative regulator of RNA polymerase II (pol II) transcription, but it has also been implicated in mRNA turnover and protein ubiquitination. We investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU induced Sml1p degradation. We show, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or methyl-methane sulfonate (MMS) treatment. Chromatin immunoprecipitation (ChIP) experiments show that TBP, pol II and Set1p recruitment to the activated RNR3 locus is defective in cells lacking NOT4. Moreover, RNR3-promoter activity is not induced by HU in these cells. Our experiments show that induction of RNR gene transcription is defective in ccr4-not mutant strains, providing an explanation for their sensitivity to HU.

Base Sequence↗
PubMed1990

Inhibition of infant glucocorticoid stress response: specific role of maternal cues.

Previous work from this laboratory has shown that passive contact with an anesthetized lactating dam eliminates the corticosteroid stress response to novelty in 12-, 16-, and 20-day-old rat pups. In the present study, we further examined some of the sensory and developmental aspects of this phenomenon. In two experiments, we asked whether inhibition of the corticosteroid stress response is related exclusively to contact with a maternal stimulus as opposed to other social stimuli. Experiment I compared the effectiveness of contact with an anesthetized dam vs, an anesthetized adult male rat. At 12 and 16 days of age, contact with an adult male was somewhat effective in reducing stress responsiveness, whereas at 20 days, contact with an adult male was entirely ineffective. At all ages, contact with an anesthetized dam was an effective inhibitor of the stress response. Experiment II compared the relative effectiveness of contact with an anesthetized dam vs. an anesthetized sibling pup. At all ages, contact with a dam was very effective, whereas contact with an anesthetized pup was entirely ineffective in inhibiting the glucocorticoid stress response. In Experiment III, the effect of maternal contact was assessed during a period of development when the biological, and presumably social, significance of the lactating-dam stimulus is changing. In this study, 20-, 24-, and 28-day-old rat pups revealed a progressive decrement in the ability of maternal contact to inhibit stress responsiveness. These findings replicate and extend our previous ones by showing that maternal stimuli play a special role in the modulation of pituitary-adrenal activity during development.

Animals↗
PubMed2002

Structural and biochemical analysis of the Obg GTP binding protein.

The Obg nucleotide binding protein family has been implicated in stress response, chromosome partitioning, replication initiation, mycelium development, and sporulation. Obg proteins are among a large group of GTP binding proteins conserved from bacteria to man. Members of the family contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain. Structural analysis of Bacillus subtilis Obg revealed respective domain architectures and how they are coupled through the putative switch elements of the C-terminal GTPase domain in apo and nucleotide-bound configurations. Biochemical analysis of bacterial and human Obg proteins combined with the structural observation of the ppGpp nucleotide within the Obg active sight suggest a potential role for ppGpp modulation of Obg function in B. subtilis.

Amino Acid Sequence↗
PubMed2005

ATR activation necessary but not sufficient for p53 induction and apoptosis in hydroxyurea-hypersensitive myeloid leukemia cells.

Hydroxyurea (HU) is a competitive inhibitor of ribonucleotide reductase that is used for the treatment of myeloproliferative disorders. HU inhibits DNA replication and induces apoptosis in a cell type-dependent manner, yet the relevant pathways that mediate apoptosis in response to this agent are not well characterized. In this study, we employed the human myeloid leukemia 1 (ML-1) cell line as a model to investigate the mechanisms of HU-induced apoptosis. Exposure of ML-1 cells to HU caused rapid cell death that was accompanied by hallmark features of apoptosis, including membrane blebbing, phosphatidylserine translocation, and caspase activation. HU-induced apoptosis required new protein synthesis, was induced by HU exposures as short as 15 min, and correlated with the accumulation of p53 and induction of the p53 target gene PUMA. p53 induction in ML-1 cells was ATR dependent and downregulation of p53 through RNAi delayed HU-induced apoptosis. HU did not induce p53 or induce apoptosis in Molt-3 leukemia cells, even though exposure to HU induced a comparable level of DNA damage and robustly activated the ATR pathway. The microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event contributes to p53 induction and apoptosis in this cell line. Our findings outline an HU-induced cell death pathway and suggest that activation of ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.

Apoptosis↗
PubMed2006

"ATR activation in response to ionizing radiation: still ATM territory".

Unrepaired DNA double-strand breaks (DSBs) are a major cause for genomic instability. Therefore, upon detection of a DSB a rapid response must be assembled to coordinate the proper repair/signaling of the lesion or the elimination of cells with unsustainable amounts of DNA damage. Three members of the PIKK family of protein kinases -ATM, ATR and DNA-PKcs- take the lead and initiate the signaling cascade emanating from DSB sites. Whereas DNA-PKcs activity seems to be restricted to the phosphorylation of targets involved in DNA repair, ATM and ATR phosphorylate a broad spectrum of cell cycle regulators and DNA repair proteins. In the canonical model, ATM and ATR are activated by two different types of lesions and signal through two independent and alternate pathways. Specifically, ATR is activated by various forms of DNA damage, including DSBs, arising at stalled replication forks ("replication stress"), and ATM is responsible for the signaling of DSBs that are not associated with the replication machinery throughout the cell cycle. Recent evidence suggests that this model might be oversimplified and that coordinated crosstalk between ATM and ATR activation routes goes on at the core of the DNA damage response.

Journal Article↗
PubMed1991

The effect of restraint stress on the kinetics, magnitude, and isotype of the humoral immune response to influenza virus infection.

The stress of physical restraint has been shown to modulate the cellular immune response during a viral infection. We have studied the effects of stress on the humoral immune response during infection by influenza virus. Restraint stress altered the kinetics of the antibody response; seroconversion in the IgG and IgA isotypes was delayed in virus-infected C57BL/6 mice subjected to repeated cycles of physical restraint. However, the magnitude and isotype of the mature antibody response were unaffected during the plateau phase; no significant differences were observed between restrained/infected and nonrestrained/infected mice. Thus, the time during infection at which the antibody response was measured was a significant variable in the study of stress-induced alterations of the host's response to a replicating viral antigen. While restraint stress did not significantly affect the magnitude or class of the humoral response, it did alter the kinetics of response.

Animals↗
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↗
PubMed1991

Infection with adeno-associated parvovirus leads to increased sensitivity of mammalian cells to stress.

Infection of the SV40-transformed Syrian hamster cells Elona with adeno-associated parvovirus type 5 (AAV-5) affects the cellular stress response. Stress-induced growth delay becomes permanent and this promotes cell death. AAV DNA is replicated in the absence of helper virus. Induction of an incomplete stress response by cycloheximide treatment still results in replication of viral DNA but not in virus-mediated growth arrest. Thus, the reactions that induce AAV DNA replication and that direct growth delay are different and replication of AAV DNA is not sufficient to induce growth arrest and cell death. Additional steps that lead to effects resembling inhibition of protein synthesis are required. The possibility that they may have their origin in the action of AAV rep proteins is discussed.

Animals↗
PubMed2006

Claspin: timing the cell cycle arrest when the genome is damaged.

DNA damage checkpoints maintain genomic integrity by delaying cell cycle progression in response to genotoxic stress and stalled replication forks. One central pathway in the checkpoint response is the ATR-Chk1 pathway, in which, upon DNA damage, ATR phosphorylates and activates the effector kinase Chk1. This process depends on the adaptor protein Claspin that bridges ATR and Chk1. Once the damage is repaired, this pathway must somehow be switched off to allow the cell to continue the cell division process, an event known as checkpoint recovery. Polo-like kinase 1 (Plk1) plays a central role during checkpoint recovery. Interestingly, the Xenopus homologue of Plk1, Plx1, is able to bind and phosphorylate Claspin, releasing it from DNA and thereby contributing to Chk1 inactivation. Moreover, it was recently demonstrated that Claspin levels are controlled by proteasomal degradation, and this is regulated by Plk1. Importantly, Plk1-mediated proteosomal degradation of Claspin appears to be essential for check-point recovery. Here we review these recent findings and discuss the mechanisms of checkpoint regulation by Claspin.

Adaptor Proteins, Signal Transducing↗
PubMed2001

ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1.

Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.

Adenoviridae↗
PubMed1996

Differential response to UV stress and DNA damage during the yeast replicative life span.

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death.

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↗
PubMed2004

p53 moves to mitochondria: a turn on the path to apoptosis.

It has been said that no matter which direction cancer research turns, the p53 tumor suppressor protein comes into view. The widespread role of p53 as a suppressor of tumor development is believed to rely on its ability to induce programmed cell death in response to stress, either the replicative stress associated with uncontrolled cellular proliferation, or the environmental stresses that accompany tumor development, such as hypoxia. For some time it has been believed that the role of p53 in inducing apoptosis in response to such stress was as a master regulator coordinating the expression of other molecules whose ultimate role was the execution of the cell. New data, however, suggest that p53 itself also has a direct role in accomplishing cell death, at the mitochondria.

Apoptosis↗
PubMed2005

Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress.

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.

Apoptosis↗