DNA Damage Checkpoint In Response Research Articles

γH2A/γH2AX Mediates DNA Damage-Specific Control of Checkpoint Signaling in Saccharomyces cerevisiae.

DNA lesions trigger DNA damage checkpoint (DDC) signaling which arrests cell cycle progression and promotes DNA damage repair. In Saccharomyces cerevisiae, phosphorylation of histone H2A (γH2A, equivalent to γH2AX in mammals) is an early chromatin mark induced by DNA damage that is recognized by a group of DDC and DNA repair factors. We find that γH2A negatively regulates the G2/M checkpoint in response to the genotoxin camptothecin, which is a DNA topoisomerase I poison. γH2A also suppresses DDC signaling induced by the DNA alkylating agent methyl methanesulfonate. These results differ from prior findings, which demonstrate positive or no roles of γH2A in DDC in response to other DNA damaging agents such as phleomycin and ionizing radiation, which suggest that γH2A has DNA damage-specific effects on DDC signaling. We also find evidence supporting the notion that γH2A regulates DDC signaling by mediating the competitive recruitment of the DDC mediator Rad9 and the DNA repair factor Rtt107 to DNA lesions. We propose that γH2A/γH2AX serves to create a dynamic balance between DDC and DNA repair that is influenced by the nature of DNA damage.

DNA polymerase delta interacting protein 3 facilitates the activation and maintenance of DNA damage checkpoint in response to replication stress.

BackgroundReplication stress response is crucial for the maintenance of a stable genome. POLDIP3 (DNA polymerase delta interacting protein 3) was initially identified as one of the DNA polymerase δ (Pol δ) interacting proteins almost 20 years ago. Using a variety of in vitro biochemical assays, we previously established that POLDIP3 is a key regulator of the enzymatic activity of Pol δ. However, the in vivo function of POLDIP3 in DNA replication and DNA damage response has been elusive.MethodsWe first generated POLDIP3 knockout (KO) cells using the CRISPR/Cas9 technology. We then investigated its biological functions in vivo using a variety of biochemical and cell biology assays.ResultsWe showed that although the POLDIP3‐KO cells manifest no pronounced defect in global DNA synthesis under nonstress conditions, they are sensitive to a variety of replication fork blockers. Intriguingly, we found that POLDIP3 plays a crucial role in the activation and maintenance of the DNA damage checkpoint in response to exogenous as well as endogenous replication stress.ConclusionOur results indicate that when the DNA replication fork is blocked, POLDIP3 can be recruited to the stalled replication fork and functions to bridge the early DNA damage checkpoint response and the later replication fork repair/restart.

Adaptation in replicative senescence: a risky business.

Cell proliferation is tightly regulated to avoid propagating DNA damage and mutations, which can lead to pathologies such as cancer. To ensure genome integrity, cells activate the DNA damage checkpoint in response to genotoxic lesions to block cell cycle progression. This surveillance mechanism provides time to repair the damage before resuming cell cycle with an intact genome. When the damage is not repaired, cells can, in some conditions, override the cell cycle arrest and proceed with proliferation, a phenomenon known as adaptation to DNA damage. A subpopulation of adapted cells might eventually survive, but only at the cost of extensive genome instability. How and in which context adaptation operates the trade-off between survival and genome stability is a fascinating question. After a brief review of the current knowledge on adaptation to DNA damage in budding yeast, we will discuss a new role of adaptation in the context of telomerase-negative cells and replicative senescence. We highlight the idea that, in all settings studied so far, survival through adaptation is a double-edged sword as it comes with increased genomic instability.

CCDC6: the identity of a protein known to be partner in fusion.

Coiled Coil Domain Containing 6 gene, CCDC6, was initially isolated as part of a tumorigenic DNA originated by the fusion of CCDC6 with the tyrosine kinase of RET receptor, following a paracentric inversion of chromosome 10. For a long time, CCDC6 has been considered as an accidental partner of the RET protooncogene, providing the promoter and the first 101 aa necessary for the constitutive activation of the oncogenic Tyrosine Kinase (TK) RET in thyroid cells. With the advent of more refined diagnostic tools and bioinformatic algorithms, an exponential growth in fusion genes discoveries has allowed the identification of CCDC6 as partner of genes other than RET in different tumor types. CCDC6 gene product has a proper role in sustaining the DNA damage checkpoints in response to DNA damage. The inactivation of CCDC6 secondary to chromosomal rearrangements or gene mutations could enhance tumor progression by impairing the apoptotic response upon the DNA damage exposure, contributing to the generation of radio- and chemoresistance. Preclinical studies indicate that the attenuation of CCDC6 in cancer, while conferring a resistance to cisplatinum, sensitizes the cancer cells to the small molecule inhibitors of Poly (ADP-ribose) polymerase (PARP1/2) with a synthetic lethal effect. Several CCDC6 mutations and gene rearrangements have been described so far in different types of cancer and CCDC6 may represent a possible predictive biomarker of tumor resistance to the conventional anticancer treatments. Nevertheless, the detection of a CCDC6 impairment in cancer patients may help to select, in future clinical trials, those patients who could benefit of PARP-inhibitors treatment alone or in combination with other treatments.

RETRACTED ARTICLE: Critical role of CCDC6 in the neoplastic growth of testicular germ cell tumors

BackgroundDNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation.CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners.In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors.MethodsTo achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses.ResultsThe analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased.ConclusionsTherefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

Role of telomere dysfunction in aging and its detection by biomarkers

Aging is a complex process that has been shown to be linked to accumulation of DNA damage. Telomere shortening represents a cell-intrinsic mechanism leading to DNA damage accumulation and activation of DNA damage checkpoints in aging cells. Activation of DNA damage checkpoints in response to telomere dysfunction results in induction of cellular senescence-a permanent cell cycle arrest. Senescence represents a tumor suppressor mechanism protecting cells from evolution of genomic instability and transformation. As a drawback, telomere shortening may also limit tissue renewal and regenerative capacity of tissues in response to aging and chronic disease. In aged organs, telomere shortening may also increase the cancer risk by initiation of chromosomal instability, loss of proliferative competition of aging stem cells, and selection of aberrant growing clones. Consequently, aged individuals are more susceptible and vulnerable to various diseases and show an increased cancer risk. Recently, proteins were discovered, which are induced by telomere dysfunction and DNA damage. It was shown that these proteins represent new biomarkers of human aging and disease. Here, we review the scientific background and experimental data on these newly discovered biomarkers.

The human ATR-mediated DNA damage checkpoint in a reconstituted system

DNA damage checkpoints are signal transduction pathways that coordinate the cell cycle with other cellular responses to DNA damage in order to maintain genomic integrity. The ATR kinase is responsible for initiating the DNA damage checkpoint in response to UV-damaged DNA and incompletely replicated DNA. We have recently established an in vitro system that recapitulates most but not all features of the human ATR-mediated DNA damage checkpoint response. With this system we have shown that damaged DNA can be a direct signal for ATR activation, and that TopBP1 specifically stimulates ATR in the presence of damaged DNA under physiological ionic strength conditions. This system provides a powerful tool to gain insight into the molecular mechanism of the ATR pathway. Here we describe preparation of the checkpoint components and our specific kinase assay in more detail.

Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells

Recently, TWIST, a basic helix-loop-helix transcription factor, is suggested to be an oncogene because of its over-expression in many types of human cancer and its positive role in promoting cell survival. The aim of this study was to investigate the role of TWIST on the growth of human epithelial cells. Using two immortalized human prostate epithelial cell lines, we demonstrated that inactivation of TWIST by small RNA technology led to the promotion of cellular senescence and growth arrest, suggesting that TWIST plays a key role in the continuous proliferation of immortalized cells. Over-expression of TWIST, in contrast, resulted in suppression of cellular senescence in response to genotoxic damage and promotion of cell proliferation with DNA damage accumulation, indicating that TWIST promotes genomic instability. In addition, we also found that the TWIST-mediated cellular senescence was regulated through its negative effect on p14(ARF) and subsequent suppression of MDM2/p53 and Chk1/2 DNA damage response pathways. Our results suggest that over-expression of TWIST results in down-regulation of p14(ARF), which leads to the impairment of DNA damage checkpoint in response to genotoxic stress. This negative effect of TWIST on DNA damage response facilitates uncontrolled cell proliferation with genomic instability and tumorigenesis in non-malignant cells.

Exonuclease-1 Deletion Impairs DNA Damage Signaling and Prolongs Lifespan of Telomere-Dysfunctional Mice

Exonuclease-1 (EXO1) mediates checkpoint induction in response to telomere dysfunction in yeast, but it is unknown whether EXO1 has similar functions in mammalian cells. Here we show that deletion of the nuclease domain of Exo1 reduces accumulation of DNA damage and DNA damage signal induction in telomere-dysfunctional mice. Exo1 deletion improved organ maintenance and lifespan of telomere-dysfunctional mice but did not increase chromosomal instability or cancer formation. Deletion of Exo1 also ameliorated the induction of DNA damage checkpoints in response to gamma-irradiation and conferred cellular resistance to 6-thioguanine-induced DNA damage. Exo1 deletion impaired upstream induction of DNA damage responses by reducing ssDNA formation and the recruitment of Replication Protein A (RPA) and ATR at DNA breaks. Together, these studies provide evidence that EXO1 contributes to DNA damage signal induction in mammalian cells, and deletion of Exo1 can prolong survival in the context of telomere dysfunction.

Mice Lacking Protein Phosphatase 5 Are Defective in Ataxia Telangiectasia Mutated (ATM)-mediated Cell Cycle Arrest

Protein phosphatase 5 (Ppp5), a tetratricopeptide repeat domain protein, has been implicated in multiple cellular functions, including cellular proliferation, migration, differentiation and survival, and cell cycle checkpoint regulation via the ataxia telangiectasia mutated/ATM and Rad3-related (ATM/ATR) signal pathway. However, the physiological functions of Ppp5 have not been reported. To confirm the role of Ppp5 in cell cycle checkpoint regulation, we generated Ppp5-deficient mice and isolated mouse embryonic fibroblast (MEF) cells from Ppp5-deficient and littermate control embryos. Although Ppp5-deficient mice can survive through embryonic development and postnatal life and MEF cells from the Ppp5-deficient mice maintain normal replication checkpoint induced by hydroxyurea, Ppp5-deficient MEF cells display a significant defect in G(2)/M DNA damage checkpoint in response to ionizing radiation (IR). To determine whether this defect in IR-induced G(2)/M checkpoint is due to altered ATM-mediated signaling, we measured ATM kinase activity and ATM-mediated downstream events. Our data demonstrated that IR-induced ATM kinase activity is attenuated in Ppp5-deficient MEFs. Phosphorylation levels of two known ATM substrates, Rad17 and Chk2, were significantly reduced in Ppp5-deficient MEFs in response to IR. Furthermore, DNA damage-induced Rad17 nuclear foci were dramatically reduced in Ppp5-deficient MEFs. These results demonstrate a direct regulatory linkage between Ppp5 and activation of the ATM-mediated G(2)/M DNA damage checkpoint pathway in vivo.

Drosophila mus301/spindle-C Encodes a Helicase With an Essential Role in Double-Strand DNA Break Repair and Meiotic Progression

mus301 was identified independently in two genetic screens, one for mutants hypersensitive to chemical mutagens and another for maternal mutants with eggshell defects. mus301 is required for the proper specification of the oocyte and for progression through meiosis in the Drosophila ovary. We have cloned mus301 and show that it is a member of the Mus308 subfamily of ATP-dependent helicases and the closest homolog of human and mouse HEL308. Functional analyses demonstrate that Mus301 is involved in chromosome segregation in meiosis and in the repair of double-strand-DNA breaks in both meiotic and mitotic cells. Most of the oogenesis defects of mus301 mutants are suppressed by mutants in the checkpoint kinase Mei41 and in MeiW68, the Spo11 homolog that is thought to generate the dsDNA breaks that initiate recombination, indicating that these phenotypes are caused by activation of the DNA damage checkpoint in response to unrepaired Mei-W68-induced dsDNA breaks. However, neither mei-W68 nor mei-41 rescue the defects in oocyte specification of mus301 mutants, suggesting that this helicase has another function in oocyte selection that is independent from its role in meiotic recombination.

Glioma stem cells promote radioresistance by preferential activation of the DNA damage response

Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.

Depletion of Chk1 Leads to Premature Activation of Cdc2-cyclin B and Mitotic Catastrophe

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.

Aberrant p53 alters DNA damage checkpoints in response to cisplatin: downregulation of CDK expression and activity.

The p53 tumor suppressor protein is a critical mediator of cell cycle arrest and apoptosis in response to genotoxic stress. Abrogation of p53 function is a major feature of tumor development and may result in a compromised DNA-damage response. In our study, we examined the effect of expressing a human p53 cDNA, encoding a histidine to leucine amino acid substitution at codon 179 (H179L), on the ability of wild-type p53-containing NIH3T3 cells to respond to treatment with the chemotherapeutic cisplatin. After 72 hr of cisplatin treatment control cells underwent apoptosis preceded by a combination of S- and G(2) arrest, as judged by flow cytometry of propidium iodide-stained cells, and TUNEL and caspase-3 assays. This correlated with increased expression of the pro-apoptotic protein Bax. In contrast, cells stably expressing H179L-p53 arrested in S-phase following cisplatin treatment, which correlated with a marked decrease in the expression of cdc2, cyclin B1 and cyclin A, and a decrease in CDK2 and cyclin A-associated kinase activity. Interestingly, H179L p53 expressing cells underwent apoptosis earlier than control cells, indicating that this aberrant p53 may enhance cisplatin chemosensitivity. These data suggest that dominant-negative p53 can influence the expression and activity of CDK complexes, thereby modifying cell behavior following cisplatin-induced genotoxicity.

Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2.

Analysis of mutants defective in meiotic chromosome pairing has uncovered a role for Caenorhabditis elegans chk-2 in initial establishment of pairing between homologous chromosomes during early meiotic prophase. chk-2 is also required for the major spatial reorganization of nuclei that normally accompanies the onset of pairing, suggesting a mechanistic coupling of these two events. Despite failures in pairing, nuclear reorganization, and crossover recombination, chk-2 mutants undergo many other aspects of meiotic chromosome morphogenesis and complete gametogenesis. Although chk-2 encodes a C. elegans ortholog of the Cds1/Chk2 checkpoint protein kinases, germ-line nuclei in chk-2 mutants are competent to arrest proliferation in response to replication inhibition and to trigger DNA damage checkpoint responses to ionizing radiation. However, chk-2 mutants are defective in triggering the pachytene DNA damage checkpoint in response to an intermediate block in the meiotic recombination pathway, suggesting that chk-2 is required either for initiation of meiotic recombination or for monitoring a specific subset of DNA damage lesions. We propose that chk-2 functions during premeiotic S phase to enable chromosomes to become competent for subsequent meiotic prophase events and/or to coordinate replication with entry into prophase.

Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G(2)/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.

The SFP1 gene product of Saccharomyces cerevisiae regulates G2/M transitions during the mitotic cell cycle and DNA-damage response.

In eukaryotic cells, checkpoint pathways arrest cell-cycle progression if a particular event has failed to complete appropriately or if an important intracellular structure is defective or damaged. Saccharomyces cerevisiae strains that lack the SFP1 gene fail to arrest at the G2 DNA-damage checkpoint in response to genomic injury, but maintain their ability to arrest at the replication and spindle-assembly checkpoints. sfp1Delta mutants are characterized by a premature entrance into mitosis during a normal (undamaged) cell cycle, while strains that overexpress Sfp1p exhibit delays in G2. Sfp1p therefore acts as a repressor of the G2/M transition, both in the normal cell cycle and in the G2 checkpoint pathway. Sfp1 is a nuclear protein with two Cys2His2 zinc-finger domains commonly found in transcription factors. We propose that Sfp1p regulates the expression of gene products involved in the G2/M transition during the mitotic cell cycle and the DNA-damage response. In support of this model, overexpression of Sfp1p induces the expression of the PDS1 gene, which is known to encode a protein that regulates the G2 checkpoint.