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At least 55 records · Page 3Linked to original sources
PubMed2006

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

The WTM genes in budding yeast amplify expression of the stress-inducible gene RNR3.

Cellular responses to DNA damage and inhibited replication are evolutionarily conserved sets of pathways that are critical to preserving genome stability. To identify new participants in these responses, we undertook a screen for regulators that, when present on a high-copy vector, alter expression of a DNA damage-inducible RNR3-lacZ reporter construct in Saccharomyces cerevisiae. From this screen we isolated a plasmid encoding two closely related paralogs, WTM1 and WTM2, that greatly increases constitutive expression of RNR3-lacZ. Moderate overexpression of both genes together, or high-level expression of WTM2 alone from a constitutive promoter, upregulates RNR3-lacZ in the absence of DNA damage. Overexpressed, tagged Wtm2p is associated with the RNR3 promoter, indicating that this effect is likely direct. Further investigation reveals that Wtm2p and Wtm1p, previously described as regulators of meiotic gene expression and transcriptional silencing, amplify transcriptional induction of RNR3 in response to replication stress and modulate expression of genes encoding other RNR subunits.

Chromatin Immunoprecipitation↗
PubMed2024

Viral hijacking of hnRNPH1 unveils a G-quadruplex-driven mechanism of stress control.

Viral genomes are enriched with G-quadruplexes (G4s), non-canonical structures formed in DNA or RNA upon assembly of four guanine stretches into stacked quartets. Because of their critical roles, G4s are potential antiviral targets, yet their function remains largely unknown. Here, we characterize the formation and functions of a conserved G4 within the polymerase coding region of orthoflaviviruses of the Flaviviridae family. Using yellow fever virus, we determine that this G4 promotes viral replication and suppresses host stress responses via interactions with hnRNPH1, a host nuclear protein involved in RNA processing. G4 binding to hnRNPH1 causes its cytoplasmic retention with subsequent impacts on G4-containing tRNA fragments (tiRNAs) involved in stress-mediated reductions in translation. As a result, these host stress responses and associated antiviral effects are impaired. These data reveal that the interplay between hnRNPH1 and both host and viral G4 targets controls the integrated stress response and viral replication.

Animals↗
PubMed2006

Methods for studying mutagenesis and checkpoints in Schizosaccharomyces pombe.

Mutations in genome caretaker genes can induce genomic instability, which are potentially early events in tumorigenesis. Cells have evolved biological processes to cope with the genomic insults. One is a multifaceted response, termed checkpoint, which is a network of signaling pathways to coordinate cell cycle transition with DNA repair, activation of transcriptional programs, and induction of tolerance of the genomic perturbations. When genomic perturbations are beyond repair, checkpoint responses can also induce apoptosis or senescence to eliminate those deleterious damaged cells. Fission yeast, Schizosaccharomyces pombe (S. pombe) has served as a valuable model organism for studies of the checkpoint signaling pathways. In this chapter, we describe methods used to analyze mutagenesis and recombinational repair induced by genomic perturbations, and methods used to detect the checkpoint responses to replication stress and DNA damage in fission yeast cells. In the first section, we present methods used to analyze the mutation rate, mutation spectra, and recombinational repair in fission yeast when replication is perturbed by either genotoxic agents or mutations in genomic caretaker gene such as DNA replication genes. In the second section, we describe methods used to examine checkpoint activation in response to chromosome replication stress and DNA damage. In the final section, we comment on how checkpoint activation regulates mutagenic synthesis by a translesion DNA polymerase in generating a mutator phenotype of small sequence alterations in cells, and how a checkpoint kinase appropriately regulates an endonuclease complex to either prevent or allow deletion of genomic sequences and recombinational repair when fission yeast cells experience genomic perturbation in order to avoid deleterious mutations and maintain cell growth.

DNA Damage↗
PubMed2005

Taming the spindle for containing the chromosomes.

Checkpoint controls are critical for coordination of cell cycle events, especially during exposure to perturbations or stresses. The DNA replication checkpoint is activated in S phase in response to replication stresses that impede fork progression. Mec1 and Rad53 are critical effectors of this control pathway; they maintain the integrity of stalled replication forks and prevent premature segregation of unreplicated chromosomes. It has long been thought that the checkpoint inhibits precocious segregation of chromosomes by preventing early onset of mitosis. However, recent evidence suggests that the replication checkpoint thwarts untimely chromosome separation not by inhibiting mitotic entry but by directly regulating spindle dynamics. These findings raise a number of issues which may require a revisit to the well-trodden territories of cell cycle regulation.

Cdc20 Proteins↗
PubMed2006

An FHA domain-mediated protein interaction network of Rad53 reveals its role in polarized cell growth.

The DNA damage checkpoint kinase Rad53 is important for the survival of budding yeast under genotoxic stresses. We performed a biochemical screen to identify proteins with specific affinity for the two Forkhead associated (FHA) domains of Rad53. The N-terminal FHA1 domain was found to coordinate a complex protein interaction network, which includes nuclear proteins involved in DNA damage checkpoints and transcriptional regulation. Unexpectedly, cytosolic proteins involved in cytokinesis, including septins, were also found as FHA1 binding proteins. Consistent with this interaction, a Rad53 mutant defective in its nuclear localization was found to localize to the bud neck. Abnormal morphology was observed in cells overexpressing the FHA1 domain and in rad53Delta cells under DNA replication stress. Further, septin Shs1 appears to have an important role in the response to DNA replication stress. Collectively, the results suggest a novel function of Rad53 in the regulation of polarized cell growth in response to DNA replication stress.

Carrier Proteins↗
PubMed2004

Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation.

Claspin is a homolog of Mrc1, a checkpoint protein required for the DNA replication checkpoint in yeast. In Xenopus, phosphorylated Claspin binds to xChk1 and regulates xChk1 activation in response to replication stress. In this study, we have shown that the human homolog of Claspin is required for resistance to multiple forms of genotoxic stress including UV, IR, and hydroxyurea. Phosphorylation of Claspin was found to depend on the ataxia telangiectasia mutated-Rad3 related (ATR) pathway. DNA damage induces the formation of a complex between Claspin and BRCA1, a second regulator of Chk1 activation. Claspin was found to control BRCA1 phosphorylation on serine 1524, a site whose phosphorylation is controlled by the ATR pathway. These results are consistent with a model in which ATR regulates Claspin phosphorylation in response to DNA damage and replication stress resulting in recruitment and phosphorylation of BRCA1. BRCA1 and Claspin then function to activate the tumor suppressor Chk1. Unexpectedly, we found that Claspin has a second, positive role in control of the cell cycle as Claspin overexpression increased cell proliferation. These results suggest that Claspin has properties of both a tumor suppressor and an oncogene.

Adaptor Proteins, Signal Transducing↗
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↗
PubMed2004

ATM is required for the cellular response to thymidine induced replication fork stress.

Genetically distinct checkpoints, activated as a consequence of either DNA replication arrest or ionizing radiation-induced DNA damage, integrate DNA repair responses into the cell cycle programme. The ataxia-telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks, whereas the related ATR is important in maintaining the integrity of the DNA replication apparatus. Here, we show that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated phosphorylation of Chk2 and NBS1 and an ATM-independent phosphorylation of Chk1 and SMC1. AT cells exposed to thymidine showed decreased viability and failed to induce homologous recombination repair (HRR). Taken together, our results implicate ATM in the HRR-mediated rescue of replication forks impaired by thymidine treatment.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2000

Transportation of goats: effects on physiological stress responses and live weight loss.

The management of food animals prior to slaughter influences both profitability and animal well-being. This experiment was conducted as a split-unit design to determine live weight shrink and stress responses in goats due to differences in stocking density during transportation and holding. A total of 150 Spanish does were transported on two different days (replicate) and held overnight (18 h) without feed in low- (LD) or high-density (HD) groups. On each day, 75 does were transported 2.5 h with floor spaces of .18 m2 and .37 m2/animal in LD (25 does) and HD (50 does) groups, respectively. The average temperatures in the trailer during transportation were 34.6 and 35 degrees C, respectively, on d 1 and 2. All animals were blood-sampled before loading (PRELOAD) and four does from each treatment were sampled immediately after loading (POSTLOAD). Animals were blood-sampled in holding pens either at 0, 1, 2, 3, 4, or 18 h after transportation (time) to assess the time course (n = 8 does per time per replicate) of stress responses. Individual animals were weighed just before loading onto a trailer and after overnight holding to assess shrinkage. Treatment or treatment x time did not have a significant effect on any of the dependent variables studied. There were significant effects of time (P < .01) on plasma cortisol, glucose, and urea nitrogen (PUN) concentrations. Time also had significant effects (P < .01) on plasma creatine kinase (CK) activity, differential leukocyte counts (neutrophils, lymphocytes, monocytes, and eosinophils), and ratio of neutrophils to lymphocytes (N:L). However, plasma leptin concentrations were not influenced by time. Cortisol concentrations increased at POSTLOAD sampling, peaked at 0 h, and decreased thereafter before spiking again at 18 h of holding. The PUN was higher at 18 h than at other time periods studied. Plasma glucose concentrations increased and remained at higher levels at 0, 1, and 2 h and began decreasing at 3 h, reaching PRELOAD levels at 18 h. Plasma CK kinase activity peaked at approximately 2 h after transportation. The N:L ratio was higher at all time periods after transportation than prior to starting the journey, indicating a prolonged effect of transportation stress on the immune system. The mean (+/- SE) shrinkage losses were 10.2 +/- .68 and 9.8 +/- .68 in HD and LD treatment groups, respectively. The results indicate that the stress responses of goats due to transportation begin decreasing within 3 h after transportation. However, prolonged holding periods without feed may increase stress responses and bring about metabolic changes.

Animals↗
PubMed2014

The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage.

Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-&#x3b1; and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.

Cell Cycle Proteins↗
PubMedDate not supplied

FHA domain-mediated DNA checkpoint regulation of Rad53.

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.

Cell Cycle Proteins↗
PubMed2006

Repeated phosphopeptide motifs in human Claspin are phosphorylated by Chk1 and mediate Claspin function.

Claspin is a checkpoint protein involved in ATR (ataxia telangiectasia mutated- and Rad3-related)-dependent Chk1 activation in Xenopus and human cells. In Xenopus, Claspin interacts with Chk1 after DNA damage through a region containing two highly conserved repeats, which becomes phosphorylated during the checkpoint response. Because this region is also conserved in human Claspin, we investigated the regulation and function of these potential phosphorylation sites in human Claspin. We found that Claspin is phosphorylated in vivo at Thr-916 in response to replication stress and UV damage. Mutation of these phosphorylation sites on Claspin inhibited Claspin-Chk1 interaction in vivo, impaired Chk1 activation, and induced premature chromatin condensation in cells, indicating a defect in replication checkpoint. In addition, we found that Thr-916 on Claspin is phosphorylated by Chk1, suggesting that Chk1 regulates Claspin during checkpoint response. These results together indicate that phosphorylation of Claspin repeats in human Claspin is important for Claspin function and the regulation of Claspin-Chk1 interaction in human cells.

Adaptor Proteins, Signal Transducing↗
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↗
PubMed2004

Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks.

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.

Acid Anhydride Hydrolases↗
PubMed2004

Damage signaling: RecQ sends an SOS to you.

The DNA helicase RecQ is required for proper induction of the SOS response to replication stress in Escherichia coli. Unwinding of stalled replication forks by RecQ family helicases in bacteria, and possibly in eukaryotes, may provide a means of damage signaling and recovering stalled replication forks.

Adenosine Triphosphatases↗
PubMed2006

Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase delta subunit, Pol31.

Saccharomyces cerevisiae DNA polymerase delta (Pol delta) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Pol delta. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Pol delta complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Pol delta. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Pol deltain vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.

Adenosine Triphosphatases↗