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PubMed2006

Detecting repair intermediates in vivo: effects of DNA damage response genes on single-stranded DNA accumulation at uncapped telomeres in budding yeast.

Single-stranded DNA (ssDNA) is an important intermediate in many DNA repair pathways. Here we describe protocols that permit the measurement of ssDNA that has arisen in the yeast genome in vivo, in response to telomere uncapping. Yeast strains defective in DNA damage response (DDR) genes can be used to infer the roles of the corresponding proteins in regulating ssDNA production and in responding to ssDNA. Using column based methods to purify yeast genomic DNA and quantitative amplification of single-stranded DNA (QAOS) it is possible to measure ssDNA at numerous single copy loci in the yeast genome. We describe how to measure ssDNA in synchronous cultures of cdc13-1 mutants, containing a temperature sensitive mutation in an essential telomere capping protein, and in asynchronous cultures of yku70Delta mutants also defective in telomere capping.

Base Sequence↗

PubMed2001

Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response.

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen showed that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.

Animals↗

PubMed2006

Herpes simplex virus type I disrupts the ATR-dependent DNA-damage response during lytic infection.

Like other DNA viruses, herpes simplex virus type 1 (HSV-1) interacts with components of the cellular response to DNA damage. For example, HSV-1 sequesters endogenous, uninduced, hyperphosphorylated RPA (replication protein A) away from viral replication compartments. RPA is a ssDNA-binding protein that signals genotoxic stress through the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway. The sequestration of endogenous hyperphosphorylated RPA away from replicating viral DNA suggests that HSV-1 prevents the normal ATR-signaling response. In this study we examine the spatial distribution of endogenous hyperphosphorylated RPA with respect to ATR, its recruitment factor, ATRIP, and the cellular dsDNA break marker, gammaH2AX, during HSV-1 infection. The accumulation of these repair factors at DNA lesions has previously been identified as an early event in signaling genotoxic stress. We show that HSV-1 infection disrupts the ATR pathway by a mechanism that prevents the recruitment of repair factors, spatially uncouples ATRIP from ATR and sequesters ATRIP and endogenous hyperphosphorylated RPA within virus-induced nuclear domains containing molecular chaperones and components of the ubiquitin proteasome. The HSV-1 immediate early protein ICP0 is sufficient to induce the redistribution of ATRIP. This is the first report that a virus can disrupt the usually tight colocalization of ATR and ATRIP.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The DNA damage response in filamentous fungi.

The mechanisms used by fungal cells to repair DNA damage have been subjects of intensive investigation for almost 50 years. As a result, the model yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae have led the way in yielding critical insights into the nature of the DNA damage response. At the same time, largely through the efforts of Etta Kafer, Hirokazu Inoue, and colleagues, a substantial collection of Aspergillus nidulans and Neurospora crassa DNA repair mutants has been identified and characterized in detail. As the analysis of these mutants continues and increasing amounts of annotated genome sequence become available, it is becoming readily apparent that the DNA damage response of filamentous fungi possesses several features that distinguish it from the model yeasts. These features are emphasized in this review, which describes the genes, regulatory networks, and processes that compose the fungal DNA damage response. Further characterization of this response will likely yield general insights that are applicable to animals and plants. Moreover, it may also become evident that the DNA damage response can be manipulated to control fungal growth.

Apoptosis↗

PubMed2005

Ionizing radiations in Caenorhabditis elegans induce poly(ADP-ribosyl)ation, a conserved DNA-damage response essential for survival.

Poly(ADP-ribosyl)ation is one of the first responses to DNA damage in mammals. Although it is involved in base excision repair, its exact role has not been ascertained yet. Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 mediate most of the poly(ADP-ribosyl)ation response in mammals and are well conserved in evolution. Their respective homologues PME-1 and PME-2 are found in the nematode Caenorhabditis elegans, a well-known genetically tractable model currently used in DNA damage response research. Here we report the functional analysis of PME-1 and PME-2 in presence of DNA damage. Worms irradiated with high doses of ionizing radiations displayed a sharp drop in their NAD(+) content immediately after treatment, and a biphasic increase in poly(ADP-ribose). The physiological importance of the poly(ADP-ribosyl)ation response was highlighted when worms were preincubated with mammalian PARP inhibitors (3AB, DHQ, PJ34) and irradiated. The embryonic survival rate of the progeny was significantly decreased in a dose-dependent manner. The inhibitor 3AB had a weak effect on embryonic survival, followed closely by DHQ. However, PJ34, a member of the phenantridinone family, was very effective even when used at low concentration (100nM). In vitro PARP assay using recombinant PME-1 and PME-2 showed a similar pattern of inhibition where 3AB and DHQ were weak inhibitors, and PJ34 a stronger one. Inhibitors affect mostly the poly(ADP-ribose) polymers elongation at high concentrations. These results suggest that poly(ADP-ribosyl)ation in response to DNA damage is an ancient and very important biochemical process protecting DNA from deleterious modification.

Animals↗

PubMed2006

The DNA damage response arouses the immune system.

Although there is considerable knowledge of how DNA damage triggers cell cycle arrest, DNA repair, and apoptosis, little was known about its potential role in immune responses. Recently, we showed that genotoxic stress and stalled DNA replication forks induce the expression of ligands for the NKG2D receptor found in natural killer cells and certain T cells, cell types that are able to attack tumor cells. Chronic activation of this response in tumor cells may contribute to immune recognition, but it also imposes a selection mechanism for immune escape and malignant progression. This unique arm of the DNA damage response may have implications for understanding therapeutic responses, many of which induce the DNA damage response, and for designing more effective regimens to treat cancer.

Animals↗

PubMed2006

BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and cancer evolution.

Germline mutations of the breast cancer associated gene 1 (BRCA1) predispose women to breast and ovarian cancers. BRCA1 is a large protein with multiple functional domains and interacts with numerous proteins that are involved in many important biological processes/pathways. Mounting evidence indicates that BRCA1 is involved in all phases of the cell cycle and regulates orderly events during cell cycle progression. BRCA1 deficiency, consequently causes abnormalities in the S-phase checkpoint, the G(2)/M checkpoint, the spindle checkpoint and centrosome duplication. The genetic instability caused by BRCA1 deficiency, however, also triggers cellular responses to DNA damage that blocks cell proliferation and induces apoptosis. Thus BRCA1 mutant cells cannot develop further into full-grown tumors unless this cellular defense is broken. Functional analysis of BRCA1 in cell cycle checkpoints, genome integrity, DNA damage response (DDR) and tumor evolution should benefit our understanding of the mechanisms underlying BRCA1 associated tumorigenesis, as well as the development of therapeutic approaches for this lethal disease.

Animals↗

PubMed2005

The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor.

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50(S/S) mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50(S/S) mice and found that Rad50(S/S) cellular attrition was strongly ATM and Chk2 dependent. The hypomorphic Mre11(ATLD1) and Nbs1(Delta)(B) alleles conferred similar rescue of Rad50(S/S)-dependent hematopoietic failure. These data indicate that the Mre11 complex activates an ATM-Chk2-dependent apoptotic pathway. We find that apoptosis and cell cycle checkpoint activation are parallel outcomes of the Mre11 complex-ATM pathway. Conversely, the Rad50(S) mutation mitigated several phenotypic features of ATM deficiency. We propose that the Rad50(S) allele is hypermorphic for DNA damage signaling, and that the resulting constitutive low-level activation of the DNA damage response accounts for the partial suppression of ATM deficiency in Rad50(S/S) Atm(-/-) mice.

ATP-Binding Cassette Transporters↗

PubMed2006

Augmented cancer resistance and DNA damage response phenotypes in PPM1D null mice.

The p53-induced serine/threonine phosphatase, protein phosphatase 1D magnesium-dependent, delta isoform (PPM1D) (or wild-type p53-induced phosphatase 1 (Wip1)), exhibits oncogenic activity in vitro and in vivo. It behaves as an oncogene in rodent fibroblast transformation assays and is amplified and overexpressed in several human tumor types. It may contribute to oncogenesis through functional inactivation of p53. Here, we show that the oncogenic function of PPM1D is associated with its phosphatase activity. While overexpressed PPM1D may be oncogenic, PPM1D null mice are resistant to spontaneous tumors over their entire lifespan. This cancer resistance may be based in part on an augmented stress response following DNA damage. PPM1D null mice treated with ionizing radiation display increased p53 protein levels and increased phosphorylation of p38 MAP kinase, p53, checkpoint kinase 1 (Chk1), and checkpoint kinase 2 (Chk2) in their tissues compared to their wild-type (WT) counterparts. Male PPM1D null mice show a modest reduction in longevity, reduced serum insulin-like growth factor 1 (IGF-1) levels, and reduced body weight compared to WT mice. The PPM1D null mouse phenotypes indicate that PPM1D has a homeostatic role in abrogating the DNA damage response and may regulate aspects of male longevity.

Aging↗

PubMed2002

EDD, the human hyperplastic discs protein, has a role in progesterone receptor coactivation and potential involvement in DNA damage response.

The ubiquitin-protein ligase EDD encodes an orthologue of the hyperplastic discs tumor suppressor gene, which has a critical role in Drosophila development. Frequent allelic imbalance at the EDD chromosomal locus in human cancers suggests a role in tumorigenesis. In addition to a HECT (homologous to E6-AP carboxyl terminus) domain, the EDD protein contains a UBR1 zinc finger motif and ubiquitin-associated domain, each of which indicates involvement in ubiquitinylation pathways. This study shows that EDD interacts with importin alpha 5 through consensus basic nuclear localization signals and is localized in cell nuclei. EDD also binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation. This activity is comparable with that of the coactivator SRC-1, but, in contrast, the interaction between EDD and PR does not appear to involve an LXXLL receptor-binding motif. EDD also binds calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein, a potential target of ubiquitin-mediated proteolysis, and an altered association is found between EDD and calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein in response to DNA damage. The data presented here demonstrate a role for EDD in PR signaling but also suggest a link to cancer through DNA damage response pathways.

Amino Acid Sequence↗

PubMed2006

Epigenetic telomere protection by Drosophila DNA damage response pathways.

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.

Alleles↗

PubMed2005

Reversal of the ATM/ATR-mediated DNA damage response by the oncogenic phosphatase PPM1D.

The eukaryotic cell has evolved a sophisticated set of cell signaling pathways that respond to DNA damage and efficiently repair that damage, protecting the cell from deleterious mutations, genomic instability, and transformation into a cancerous state. The ATM and ATR serine/threonine kinases are key sensors and transducers of DNA damage signals through phosphorylation of an array of signaling molecules that mediate all aspects of the DNA damage response, including enforcement of cell cycle checkpoints and direct repair of damaged DNA. We have shown that a type 2C serine/threonine phosphatase, PPM1D (or Wip1), can reverse the phosphorylation status of ATM/ATR-phosphorylated proteins p53 and Chk1. This dephosphorylation of p53 and Chk1 by PPM1D may result in reduced functional activities and is accompanied by suppression of DNA damage-induced cell cycle checkpoints and some aspects of DNA repair. Because PPM1D is transcriptionally activated by p53 in response to DNA damage, PPM1D may serve as a critical component of a p53 negative feedback regulatory loop since it now appears that PPM1D can inhibit p53 activity by at least four different molecular mechanisms. This may explain why PPM1D is amplified and overexpressed in a subset of human breast cancers that invariably retain wild type p53 alleles. We hypothesize that PPM1D is a homeostatic regulator of the DNA damage response that returns the cell to a more normal unstressed state following repair of the damage.

Alleles↗

PubMed2006

Altered DNA damage response in Caenorhabditis elegans with impaired poly(ADP-ribose) glycohydrolases genes expression.

Poly(ADP-ribosyl)ation is one of the first cellular responses induced by DNA damage. Poly(ADP-ribose) is rapidly synthesized by nick-sensor poly(ADP-ribose) polymerases, which facilitate DNA repair enzymes to process DNA damage. ADP-ribose polymers are rapidly catabolized into free ADP-ribose units by poly(ADP-ribose) glycohydrolase (PARG). The metabolism of poly(ADP-ribose) is a well-defined biochemical process for which a physiological role in animals is just beginning to emerge. Two Caenorhabditis elegans PARGs, PME-3 and PME-4, have been cloned by our group. The pme-3 gene encodes an enzyme of 89kDa having less than 18% overall identity with human PARG but 42% identity with the PARG signature motif. The pme-4 gene codes for a PARG of 55kDa with approximately 22% overall identity with human PARG and 40% identity with the PARG signature motif. Two alternatively spliced forms of PME-3 were identified with an SL1 splice leader on both forms of the mRNA and were found to be expressed throughout the worm's life cycle. Similarly, pme-4 was shown to be expressed in all developmental stages of the worm. Recombinant enzymes that were expressed in bacteria displayed a PARG activity that may partly account for the PARG activity measured in the total worm extract. Reporter gene analysis of pme-3 and pme-4 using a GFP fusion construct showed that pme-3 and pme-4 are mainly expressed in nerve cells. PME-3 was shown to be nuclear while PME-4 localized to the cytoplasm. Worms with pme-3 and pme-4 expression knocked-down by RNAi showed a significant sensitivity toward ionizing radiations. Taken together, these data provide evidence for a physiological role for PARGs in DNA damage response and survival. It also shows that PARGs are evolutionarily conserved enzymes and that they are part of an ancient cellular response to DNA damage.

Amino Acid Sequence↗

PubMed2002

Over 1000 genes are involved in the DNA damage response of Escherichia coli.

Changes in gene expression after treatment of Escherichia coli cultures with mitomycin C were assessed using gene array technology. Unexpectedly, a large number of genes (nearly 30% of all genes) displayed significant changes in their expression level. Analysis and classification of expression profiles of the corresponding genes allowed us to assign this large number of genes into one or two dozen small clusters of genes with similar expression profiles. This assignment allowed us to describe systematically the changes in the level of gene expression in response to DNA damage. Among the damage-induced genes, more than 100 are novel. From those genes involved in DNA metabolism that have not previously been shown to be induced by DNA damage, the mutS gene involved in mismatch repair is especially noteworthy. In addition to the SOS response, we observed the induction of other stress response pathways, such as those of oxidative stress and osmotic protection. Among the genes that are downregulated in response to DNA damage are numerous protein biosynthesis genes. Analysis of the gene expression data highlighted the essential involvement of sigma(s)-regulated genes and the general stress response network in the response to DNA damage.

Bacterial Proteins↗

PubMed2005

Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression.

Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1m/m) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1m/m mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1m/m mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1m/m mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression.

Animals↗

PubMed2000

Functional link of BRCA1 and ataxia telangiectasia gene product in DNA damage response.

BRCA1 encodes a familial breast cancer suppressor that has a critical role in cellular responses to DNA damage. Mouse cells deficient for Brca1 show genetic instability, defective G2-M checkpoint control and reduced homologous recombination. BRCA1 also directly interacts with proteins of the DNA repair machinery and regulates expression of both the p21 and GADD45 genes. However, it remains unclear how DNA damage signals are transmitted to modulate the repair function of BRCA1. Here we show that the BRCA1-associated protein CtIP becomes hyperphosphorylated and dissociated from BRCA1 upon ionizing radiation. This phosphorylation event requires the protein kinase (ATM) that is mutated in the disease ataxia telangiectasia. ATM phosphorylates CtIP at serine residues 664 and 745, and mutation of these sites to alanine abrogates the dissociation of BRCA1 from CtIP, resulting in persistent repression of BRCA1-dependent induction of GADD45 upon ionizing radiation. We conclude that ATM, by phosphorylating CtIP upon ionizing radiation, may modulate BRCA1-mediated regulation of the DNA damage-response GADD45 gene, thus providing a potential link between ATM deficiency and breast cancer.

Animals↗

PubMed2006

[Relationship between abnormalities of genes involved in DNA damage responses and malignant tumors/autoimmune diseases].

The maintenance of genomic stability is an essential cellular function for a variety of well-coordinated regulation of biological activities of organisms, and a failure in its function results in the accumulation of mutations and/or abnormality in the induction of apoptosis, eventually leading to onsets of various diseases, including malignant tumors. DNA damage responses, in particular cell-cycle checkpoint regulation, play important roles in maintaining genomic integrity. In response to DNA damages induced by gamma-irradiation, ultraviolet irradiation, various chemicals, or reactive oxygen species (ROS), intrinsic cell-cycle checkpoint machinery is rapidly activated to arrest cells at particular cell-cycle points, and during cell-cycle checkpoint arrest cells may try to repair damaged DNAs, and then re-start cell-cycle upon the completion of DNA repair. Alternatively, if the extents of DNA damage overwhelm the capacity of the cellular repair machinery, cells may undergo apoptosis to prevent the accumulation of mutations within the organisms. In this article, we will first explain about our current view of DNA damage responses, in particular cell-cycle checkpoint regulation, and summarize our knowledge of the relationships between abnormalities of genes involved in DNA damage responses and malignant tumors, including hematopoietic malignancies. We will also discuss a possible implication of DNA damage responses in autoimmune diseases, such as rheumatoid arthritis.

Apoptosis↗

PubMed2007

DNA damage responses in neural cells: Focus on the telomere.

Postmitotic neurons must survive for the entire life of the organism and be able to respond adaptively to adverse conditions of oxidative and genotoxic stress. Unrepaired DNA damage can trigger apoptosis of neurons which is typically mediated by the ataxia telangiectasia mutated (ATM)-p53 pathway. As in all mammalian cells, telomeres in neurons consist of TTAGGG DNA repeats and several associated proteins that form a nucleoprotein complex that prevents chromosome ends from being recognized as double strand breaks. Proteins that stabilize telomeres include TRF1 and TRF2, and proteins known to play important roles in DNA damage responses and DNA repair including ATM, Werner and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). We have been performing studies of developing and adult neurons aimed at understanding the effects of global and telomere-directed DNA damage responses in neuronal plasticity and survival in the contexts of aging and neurodegenerative disorders. Deficits in specific DNA repair proteins, including DNA-PKcs and uracil DNA glycosylase (UDG), render neurons vulnerable to adverse conditions of relevance to the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and stroke. Similarly, early postmitotic neurons with reduced telomerase activity exhibit accentuated responses to DNA damage and are prone to apoptosis demonstrating a pivotal role for telomere maintenance in both mitotic cells and postmitotic neurons. Our recent findings suggest key roles for TRF2 in regulating the differentiation and survival of neurons. TRF2 affects cell survival and differentiation by modulating DNA damage pathways, and gene expression. A better understanding of the molecular mechanisms by which neurons respond to global and telomere-specific DNA damage may reveal novel strategies for prevention and treatment of neurodegenerative disorders. Indeed, work in this and other laboratories has shown that dietary folic acid can protect neurons against Alzheimer's disease by keeping homocysteine levels low and thereby minimizing the misincorporation of uracil into DNA in neurons.

Animals↗