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Sample records for damage response dna

  1. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  2. Deciphering the DNA Damage Response.

    PubMed

    Haber, James E

    2015-09-10

    This year's Albert Lasker Basic Medical Research Award honors Evelyn Witkin and Stephen J. Elledge, two pioneers in elucidating the DNA damage response, whose contributions span more than 40 years. PMID:26359974

  3. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  4. Targeting DNA damage response in cancer therapy

    PubMed Central

    Hosoya, Noriko; Miyagawa, Kiyoshi

    2014-01-01

    Cancer chemotherapy and radiotherapy are designed to kill cancer cells mostly by inducing DNA damage. DNA damage is normally recognized and repaired by the intrinsic DNA damage response machinery. If the damaged lesions are successfully repaired, the cells will survive. In order to specifically and effectively kill cancer cells by therapies that induce DNA damage, it is important to take advantage of specific abnormalities in the DNA damage response machinery that are present in cancer cells but not in normal cells. Such properties of cancer cells can provide biomarkers or targets for sensitization. For example, defects or upregulation of the specific pathways that recognize or repair specific types of DNA damage can serve as biomarkers of favorable or poor response to therapies that induce such types of DNA damage. Inhibition of a DNA damage response pathway may enhance the therapeutic effects in combination with the DNA-damaging agents. Moreover, it may also be useful as a monotherapy when it achieves synthetic lethality, in which inhibition of a complementary DNA damage response pathway selectively kills cancer cells that have a defect in a particular DNA repair pathway. The most striking application of this strategy is the treatment of cancers deficient in homologous recombination by poly(ADP-ribose) polymerase inhibitors. In this review, we describe the impact of targeting the cancer-specific aberrations in the DNA damage response by explaining how these treatment strategies are currently being evaluated in preclinical or clinical trials. PMID:24484288

  5. MicroRNA response to DNA damage

    PubMed Central

    Wan, Guohui; Mathur, Rohit; Hu, Xiaoxiao; Zhang, Xinna; Lu, Xiongbin

    2011-01-01

    Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution, but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways, composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that microRNAs (miRNAs) play a critical role in regulation of DNA damage response. Here, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage. PMID:21741842

  6. Cellular responses to environmental DNA damage

    SciTech Connect

    Not Available

    1994-08-01

    This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

  7. Triplex-Induced DNA Damage Response

    PubMed Central

    Rogers, Faye A.; Tiwari, Meetu Kaushik

    2013-01-01

    Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage. PMID:24348211

  8. Ubiquitylation, neddylation and the DNA damage response

    PubMed Central

    Brown, Jessica S.; Jackson, Stephen P.

    2015-01-01

    Failure of accurate DNA damage sensing and repair mechanisms manifests as a variety of human diseases, including neurodegenerative disorders, immunodeficiency, infertility and cancer. The accuracy and efficiency of DNA damage detection and repair, collectively termed the DNA damage response (DDR), requires the recruitment and subsequent post-translational modification (PTM) of a complex network of proteins. Ubiquitin and the ubiquitin-like protein (UBL) SUMO have established roles in regulating the cellular response to DNA double-strand breaks (DSBs). A role for other UBLs, such as NEDD8, is also now emerging. This article provides an overview of the DDR, discusses our current understanding of the process and function of PTM by ubiquitin and NEDD8, and reviews the literature surrounding the role of ubiquitylation and neddylation in DNA repair processes, focusing particularly on DNA DSB repair. PMID:25833379

  9. Historical perspective on the DNA damage response.

    PubMed

    Hanawalt, Philip C

    2015-12-01

    The DNA damage response (DDR) has been broadly defined as a complex network of cellular pathways that cooperate to sense and repair lesions in DNA. Multiple types of DNA damage, some natural DNA sequences, nucleotide pool deficiencies and collisions with transcription complexes can cause replication arrest to elicit the DDR. However, in practice, the term DDR as applied to eukaryotic/mammalian cells often refers more specifically to pathways involving the activation of the ATM (ataxia-telangiectasia mutated) and ATR (ATM-Rad3-related) kinases in response to double-strand breaks or arrested replication forks, respectively. Nevertheless, there are distinct responses to particular types of DNA damage that do not involve ATM or ATR. In addition, some of the aberrations that cause replication arrest and elicit the DDR cannot be categorized as direct DNA damage. These include nucleotide pool deficiencies, nucleotide sequences that can adopt non-canonical DNA structures, and collisions between replication forks and transcription complexes. The response to these aberrations can be called the genomic stress response (GSR), a term that is meant to encompass the sensing of all types of DNA aberrations together with the mechanisms involved in coping with them. In addition to fully functional cells, the consequences of processing genomic aberrations may include mutagenesis, genomic rearrangements and lethality. PMID:26507443

  10. DNA damage responses in mammalian oocytes.

    PubMed

    Collins, Josie K; Jones, Keith T

    2016-07-01

    DNA damage acquired during meiosis can lead to infertility and miscarriage. Hence, it should be important for an oocyte to be able to detect and respond to such events in order to make a healthy egg. Here, the strategies taken by oocytes during their stages of growth to respond to DNA damaging events are reviewed. In particular, recent evidence of a novel pathway in fully grown oocytes helps prevent the formation of mature eggs with DNA damage. It has been found that fully grown germinal vesicle stage oocytes that have been DNA damaged do not arrest at this point in meiosis, but instead undergo meiotic resumption and stall during the first meiotic division. The Spindle Assembly Checkpoint, which is a well-known mitotic pathway employed by somatic cells to monitor chromosome attachment to spindle microtubules, appears to be utilised by oocytes also to respond to DNA damage. As such maturing oocytes are arrested at metaphase I due to an active Spindle Assembly Checkpoint. This is surprising given this checkpoint has been previously studied in oocytes and considered to be weak and ineffectual because of its poor ability to be activated in response to microtubule attachment errors. Therefore, the involvement of the Spindle Assembly Checkpoint in DNA damage responses of mature oocytes during meiosis I uncovers a novel second function for this ubiquitous cellular checkpoint. PMID:27069010

  11. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  12. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  13. Epigenome Maintenance in Response to DNA Damage.

    PubMed

    Dabin, Juliette; Fortuny, Anna; Polo, Sophie E

    2016-06-01

    Organism viability relies on the stable maintenance of specific chromatin landscapes, established during development, that shape cell functions and identities by driving distinct gene expression programs. Yet epigenome maintenance is challenged during transcription, replication, and repair of DNA damage, all of which elicit dynamic changes in chromatin organization. Here, we review recent advances that have shed light on the specialized mechanisms contributing to the restoration of epigenome structure and function after DNA damage in the mammalian cell nucleus. By drawing a parallel with epigenome maintenance during replication, we explore emerging concepts and highlight open issues in this rapidly growing field. In particular, we present our current knowledge of molecular players that support the coordinated maintenance of genome and epigenome integrity in response to DNA damage, and we highlight how nuclear organization impacts genome stability. Finally, we discuss possible functional implications of epigenome plasticity in response to genotoxic stress. PMID:27259203

  14. The RNA Response to DNA Damage.

    PubMed

    Giono, Luciana E; Nieto Moreno, Nicolás; Cambindo Botto, Adrián E; Dujardin, Gwendal; Muñoz, Manuel J; Kornblihtt, Alberto R

    2016-06-19

    Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals, and executes multiple programs including DNA repair, cell cycle arrest, senescence, and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation, and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields. PMID:26979557

  15. Molecular mechanisms involved in initiation of the DNA damage response

    PubMed Central

    Barnum, Kevin J; O’Connell, Matthew J

    2015-01-01

    DNA is subject to a wide variety of damage. In order to maintain genomic integrity, cells must respond to this damage by activating repair and cell cycle checkpoint pathways. The initiating events in the DNA damage response entail recognition of the lesion and the assembly of DNA damage response complexes at the DNA. Here, we review what is known about these processes for various DNA damage pathways. PMID:27308403

  16. DNA Damage Response and Immune Defense: Links and Mechanisms

    PubMed Central

    Nakad, Rania; Schumacher, Björn

    2016-01-01

    DNA damage plays a causal role in numerous human pathologies including cancer, premature aging, and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR) orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signaling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signaling. We highlight evidence gained into (i) which molecular and cellular pathways of DDR activate immune signaling, (ii) how DNA damage drives chronic inflammation, and (iii) how chronic inflammation causes DNA damage and pathology in humans. PMID:27555866

  17. Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress.

    PubMed

    Dong, Daoyin; Yu, Jingwen; Wu, Yanqing; Fu, Noah; Villela, Natalia Arias; Yang, Peixin

    2015-11-13

    DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy. PMID:26427872

  18. Pseudo-DNA damage response in senescent cells

    PubMed Central

    Pospelova, Tatyana V.; Demidenko, Zoya N.; Bukreeva, Elena I.; Pospelov, Valery A.; Gudkov, Andrei V.; Blagosklonny, Mikhail V.

    2016-01-01

    Cellular senescence is currently viewed as a response to DNA damage. In this report, we showed that non-damaging agents such as sodium butyrate-induced p21 and ectopic expression of either p21 or p16 cause cellular senescence without detectable DNA breaks. Nevertheless, senescent cells displayed components of DNA damage response (DDR) such as γH2AX foci and uniform nuclear staining for p-ATM. Importantly, there was no accumulation of 53BP1 in γH2AX foci of senescent cells. Consistently, comet assay failed to detect DNA damage. Rapamycin, an inhibitor of mTOR, which was shown to suppress cellular senescence, decreased γH2AX foci formation. Thus, cellular senescence leads to activation of atypical DDR without detectable DNA damage. Pseudo-DDR may be a marker of general over-activation of senescent cells. PMID:19946210

  19. The DNA damage response: the omics era and its impact

    PubMed Central

    Derks, Kasper W.J.; Hoeijmakers, Jan H.J.; Pothof, Joris

    2014-01-01

    The emergence of high density technologies monitoring the genome, transcriptome and proteome in relation to genotoxic stress have tremendously enhanced our knowledge on global responses and dynamics in the DNA damage response, including its relation with cancer and aging. Moreover, ‘-omics’ technologies identified many novel factors, their post-translational modifications, pathways and global responses in the cellular response to DNA damage. Based on omics, it is currently estimated that thousands of gene(product)s participate in the DNA damage response, recognizing complex networks that determine cell fate after damage to the most precious cellular molecule, DNA. The development of next generation sequencing technology and associated specialized protocols can quantitatively monitor RNA and DNA at unprecedented single nucleotide resolution. In this review we will discuss the contribution of omics technologies and in particular next generation sequencing to our understanding of the DNA damage response and the future prospective of next generation sequencing, its single cell application and omics dataset integration in unraveling intricate DNA damage signaling networks. PMID:24794401

  20. Parvovirus Diversity and DNA Damage Responses

    PubMed Central

    Cotmore, Susan F.; Tattersall, Peter

    2013-01-01

    Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral “rolling hairpin” DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ∼260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell’s entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment. PMID:23293137

  1. Parvovirus diversity and DNA damage responses.

    PubMed

    Cotmore, Susan F; Tattersall, Peter

    2013-02-01

    Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral "rolling hairpin" DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ~260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell's entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment. PMID:23293137

  2. Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

    PubMed Central

    Li, Na; Parrish, Marcus; Chan, Tze Khee; Yin, Lu; Rai, Prashant; Yoshiyuki, Yamada; Abolhassani, Nona; Tan, Kong Bing; Kiraly, Orsolya; Chow, Vincent TK; Engelward, Bevin P.

    2016-01-01

    Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe Influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of two weeks in vivo. We show that influenza induces DNA damage both when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage, persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67 positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DSB repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome. PMID:25809161

  3. Chromatin perturbations during the DNA damage response in higher eukaryotes.

    PubMed

    Bakkenist, Christopher J; Kastan, Michael B

    2015-12-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response

  4. Chromatin perturbations during the DNA damage response in higher eukaryotes

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response

  5. Leishmania major and Trypanosoma cruzi present distinct DNA damage responses.

    PubMed

    Garcia, Juliana B F; Rocha, João P Vieira da; Costa-Silva, Héllida M; Alves, Ceres L; Machado, Carlos R; Cruz, Angela K

    2016-05-01

    Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification. PMID:27188657

  6. Is DNA Damage Response Ready for Action Anywhere?

    PubMed Central

    Terradas, Mariona; Martín, Marta; Hernández, Laia; Tusell, Laura; Genescà, Anna

    2012-01-01

    Organisms are continuously exposed to DNA damaging agents, consequently, cells have developed an intricate system known as the DNA damage response (DDR) in order to detect and repair DNA lesions. This response has to be rapid and accurate in order to keep genome integrity. It has been observed that the condensation state of chromatin hinders a proper DDR. However, the condensation state of chromatin is not the only barrier to DDR. In this review, we have collected data regarding the presence of DDR factors on micronuclear DNA lesions that indicate that micronuclei are almost incapable of generating an effective DDR because of defects in their nuclear envelope. Finally, considering the recent observations about the reincorporation of micronuclei to the main bulk of chromosomes, we suggest that, under certain circumstances, micronuclei carrying DNA damage might be a source of chromosome instability. PMID:23109871

  7. NBS1 and multiple regulations of DNA damage response

    PubMed Central

    Komatsu, Kenshi

    2016-01-01

    DNA damage response is finely tuned, with several pathways including those for DNA repair, chromatin remodeling and cell cycle checkpoint, although most studies to date have focused on single pathways. Genetic diseases characterized by genome instability have provided novel insights into the underlying mechanisms of DNA damage response. NBS1, a protein responsible for the radiation-sensitive autosomal recessive disorder Nijmegen breakage syndrome, is one of the first factors to accumulate at sites of DNA double-strand breaks (DSBs). NBS1 binds to at least five key proteins, including ATM, RPA, MRE11, RAD18 and RNF20, in the conserved regions within a limited span of the C terminus, functioning in the regulation of chromatin remodeling, cell cycle checkpoint and DNA repair in response to DSBs. In this article, we reviewed the functions of these binding proteins and their comprehensive association with NBS1. PMID:27068998

  8. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  9. Regulation of the DNA damage response by ubiquitin conjugation

    PubMed Central

    Brinkmann, Kerstin; Schell, Michael; Hoppe, Thorsten; Kashkar, Hamid

    2015-01-01

    In response to DNA damage, cells activate a highly conserved and complex kinase-based signaling network, commonly referred to as the DNA damage response (DDR), to safeguard genomic integrity. The DDR consists of a set of tightly regulated events, including detection of DNA damage, accumulation of DNA repair factors at the site of damage, and finally physical repair of the lesion. Upon overwhelming damage the DDR provokes detrimental cellular actions by involving the apoptotic machinery and inducing a coordinated demise of the damaged cells (DNA damage-induced apoptosis, DDIA). These diverse actions involve transcriptional activation of several genes that govern the DDR. Moreover, recent observations highlighted the role of ubiquitylation in orchestrating the DDR, providing a dynamic cellular regulatory circuit helping to guarantee genomic stability and cellular homeostasis (Popovic et al., 2014). One of the hallmarks of human cancer is genomic instability (Hanahan and Weinberg, 2011). Not surprisingly, deregulation of the DDR can lead to human diseases, including cancer, and can induce resistance to genotoxic anti-cancer therapy (Lord and Ashworth, 2012). Here, we summarize the role of ubiquitin-signaling in the DDR with special emphasis on its role in cancer and highlight the therapeutic value of the ubiquitin-conjugation machinery as a target in anti-cancer treatment strategy. PMID:25806049

  10. Decoupling of DNA damage response signaling from DNA damages underlies temozolomide resistance in glioblastoma cells☆

    PubMed Central

    Cui, Bo; Johnson, Stewart P.; Bullock, Nancy; Ali-Osman, Francis; Bigner, Darell D.; Friedman, Henry S.

    2010-01-01

    Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor in adults. Current therapy includes surgery, radiation and chemotherapy with temozolomide (TMZ). Major determinants of clinical response to TMZ include methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter and mismatch repair (MMR) status. Though the MGMT promoter is methylated in 45% of cases, for the first nine months of follow-up, TMZ does not change survival outcome. Furthermore, MMR deficiency makes little contribution to clinical resistance, suggesting that there exist unrecognized mechanisms of resistance. We generated paired GBM cell lines whose resistance was attributed to neither MGMT nor MMR. We show that, responding to TMZ, these cells exhibit a decoupling of DNA damage response (DDR) from ongoing DNA damages. They display methylation-resistant synthesis in which ongoing DNA synthesis is not inhibited. They are also defective in the activation of the S and G2 phase checkpoint. DDR proteins ATM, Chk2, MDC1, NBS1 and gammaH2AX also fail to form discrete foci. These results demonstrate that failure of DDR may play an active role in chemoresistance to TMZ. DNA damages by TMZ are repaired by MMR proteins in a futile, reiterative process, which activates DDR signaling network that ultimately leads to the onset of cell death. GBM cells may survive genetic insults in the absence of DDR. We anticipate that our findings will lead to more studies that seek to further define the role of DDR in ultimately determining the fate of a tumor cell in response to TMZ and other DNA methylators. PMID:23554659

  11. Quality control mechanisms in cellular and systemic DNA damage responses

    PubMed Central

    Ermolaeva, Maria A.; Dakhovnik, Alexander; Schumacher, Björn

    2016-01-01

    The maintenance of the genome is of pivotal importance for the functional integrity of cells and tissues. The gradual accumulation of DNA damage is thought to contribute to the functional decline of tissues and organs with ageing. Defects in multiple genome maintenance systems cause human disorders characterized by cancer susceptibility, developmental failure, and premature ageing. The complex pathological consequences of genome instability are insufficiently explained by cell-autonomous DNA damage responses (DDR) alone. Quality control pathways play an important role in DNA repair and cellular DDR pathways. Recent years have revealed non-cell autonomous effects of DNA damage that impact the physiological adaptations during ageing. We will discuss the role of quality assurance pathways in cell-autonomous and systemic responses to genome instability. PMID:25560147

  12. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  13. DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors

    PubMed Central

    Francia, Sofia; Cabrini, Matteo; Matti, Valentina; Oldani, Amanda; d'Adda di Fagagna, Fabrizio

    2016-01-01

    ABSTRACT The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling. PMID:26906421

  14. DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors.

    PubMed

    Francia, Sofia; Cabrini, Matteo; Matti, Valentina; Oldani, Amanda; d'Adda di Fagagna, Fabrizio

    2016-04-01

    The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling. PMID:26906421

  15. Roles of RNA-Binding Proteins in DNA Damage Response

    PubMed Central

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  16. Roles of RNA-Binding Proteins in DNA Damage Response.

    PubMed

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the

  17. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  18. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  19. At a crossroads: human DNA tumor viruses and the host DNA damage response.

    PubMed

    Nikitin, Pavel A; Luftig, Micah A

    2011-07-01

    Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection. PMID:21927617

  20. Initiation of DNA damage responses through XPG-related nucleases.

    PubMed

    Kuntz, Karen; O'Connell, Matthew J

    2013-01-23

    Lesion-specific enzymes repair different forms of DNA damage, yet all lesions elicit the same checkpoint response. The common intermediate required to mount a checkpoint response is thought to be single-stranded DNA (ssDNA), coated by replication protein A (RPA) and containing a primer-template junction. To identify factors important for initiating the checkpoint response, we screened for genes that, when overexpressed, could amplify a checkpoint signal to a weak allele of chk1 in fission yeast. We identified Ast1, a novel member of the XPG-related family of endo/exonucleases. Ast1 promotes checkpoint activation caused by the absence of the other XPG-related nucleases, Exo1 and Rad2, the homologue of Fen1. Each nuclease is recruited to DSBs, and promotes the formation of ssDNA for checkpoint activation and recombinational repair. For Rad2 and Exo1, this is independent of their S-phase role in Okazaki fragment processing. This XPG-related pathway is distinct from MRN-dependent responses, and each enzyme is critical for damage resistance in MRN mutants. Thus, multiple nucleases collaborate to initiate DNA damage responses, highlighting the importance of these responses to cellular fitness. PMID:23211746

  1. Involvement of DNA Damage Response Pathways in Hepatocellular Carcinoma

    PubMed Central

    Yang, Sheau-Fang; Wei, Ren-Jie; Shiue, Yow-Ling; Wang, Shen-Nien

    2014-01-01

    Hepatocellular carcinoma (HCC) has been known as one of the most lethal human malignancies, due to the difficulty of early detection, chemoresistance, and radioresistance, and is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. Its development has been closely associated with multiple risk factors, including hepatitis B and C virus infection, alcohol consumption, obesity, and diet contamination. Genetic alterations and genomic instability, probably resulted from unrepaired DNA lesions, are increasingly recognized as a common feature of human HCC. Dysregulation of DNA damage repair and signaling to cell cycle checkpoints, known as the DNA damage response (DDR), is associated with a predisposition to cancer and affects responses to DNA-damaging anticancer therapy. It has been demonstrated that various HCC-associated risk factors are able to promote DNA damages, formation of DNA adducts, and chromosomal aberrations. Hence, alterations in the DDR pathways may accumulate these lesions to trigger hepatocarcinogenesis and also to facilitate advanced HCC progression. This review collects some of the most known information about the link between HCC-associated risk factors and DDR pathways in HCC. Hopefully, the review will remind the researchers and clinicians of further characterizing and validating the roles of these DDR pathways in HCC. PMID:24877058

  2. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

    PubMed Central

    Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

    2015-01-01

    Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

  3. DNA damage response during mouse oocyte maturation.

    PubMed

    Mayer, Alexandra; Baran, Vladimir; Sakakibara, Yogo; Brzakova, Adela; Ferencova, Ivana; Motlik, Jan; Kitajima, Tomoya S; Schultz, Richard M; Solc, Petr

    2016-01-01

    Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation. PMID:26745237

  4. Pluripotent Stem Cells and DNA Damage Response to Ionizing Radiations.

    PubMed

    Mujoo, Kalpana; Butler, E Brian; Pandita, Raj K; Hunt, Clayton R; Pandita, Tej K

    2016-07-01

    Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells, are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimal risk of rejection. Radiation-induced DNA damage, ultraviolet light, genotoxic stress and other intrinsic and extrinsic factors triggers a series of biochemical reactions known as DNA damage response. To maintain genomic stability and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling, a contrast to somatic cells. Stem cell transplantation may protect against radiation-induced late effects. In particular, this review focuses on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences. PMID:27332952

  5. Pluripotent stem cells and DNA damage response to ionizing radiations

    PubMed Central

    Mujoo, Kalpana; Butler, E. Brian; Pandita, Raj K.; Hunt, Clayton R.; Pandita, Tej K.

    2016-01-01

    Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes, and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimum risks of rejection. DNA damage induced by ionizing radiation (IR), ultraviolet (UV) light, genotoxic stress, and other intrinsic and extrinsic factors trigger a series of biochemical reactions termed as DNA damage response (DDR). In order to maintain genomic stability, and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling – a contrast to somatic cells. Stem cell transplantation may over come the late effects related to radiation. This review will particularly focus on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences. PMID:27332952

  6. Diseases Associated with Defective Responses to DNA Damage

    PubMed Central

    O’Driscoll, Mark

    2012-01-01

    Within the last decade, multiple novel congenital human disorders have been described with genetic defects in known and/or novel components of several well-known DNA repair and damage response pathways. Examples include disorders of impaired nucleotide excision repair, DNA double-strand and single-strand break repair, as well as compromised DNA damage-induced signal transduction including phosphorylation and ubiquitination. These conditions further reinforce the importance of multiple genome stability pathways for health and development in humans. Furthermore, these conditions inform our knowledge of the biology of the mechanics of genome stability and in some cases provide potential routes to help exploit these pathways therapeutically. Here, I will review a selection of these exciting findings from the perspective of the disorders themselves, describing how they were identified, how genotype informs phenotype, and how these defects contribute to our growing understanding of genome stability pathways. PMID:23209155

  7. Modeling the Study of DNA Damage Responses in Mice

    PubMed Central

    Specks, Julia; Nieto-Soler, Maria; Lopez-Contreras, Andres J; Fernandez-Capetillo, Oscar

    2016-01-01

    Summary Damaged DNA has a profound impact on mammalian health and overall survival. In addition to being the source of mutations that initiate cancer, the accumulation of toxic amounts of DNA damage can cause severe developmental diseases and accelerate ageing. Therefore, understanding how cells respond to DNA damage has become one of the most intense areas of biomedical research in the recent years. However, whereas most mechanistic studies derive from in vitro or in cellulo work, the impact of a given mutation on a living organism is largely unpredictable. For instance, why BRCA1 mutations preferentially lead to breast cancer whereas mutations compromising mismatch repair drive colon cancer is still not understood. In this context, evaluating the specific physiological impact of mutations that compromise genome integrity has become crucial for a better dimensioning of our knowledge. We here describe the various technologies that can be used for modeling mutations in mice, and provide a review of the genes and pathways that have been modeled so far in the context of DNA damage responses. PMID:25636482

  8. Dissection of DNA Damage Responses Using Multiconditional Genetic Interaction Maps

    PubMed Central

    Guénolé, Aude; Srivas, Rohith; Vreeken, Kees; Wang, Ze Zhong; Wang, Shuyi; Krogan, Nevan J.; Ideker, Trey; van Attikum, Haico

    2013-01-01

    SUMMARY To protect the genome, cells have evolved a diverse set of pathways designed to sense, signal, and repair multiple types of DNA damage. To assess the degree of coordination and crosstalk among these pathways, we systematically mapped changes in the cell's genetic network across a panel of different DNA-damaging agents, resulting in ~1,800,000 differential measurements. Each agent was associated with a distinct interaction pattern, which, unlike single-mutant phenotypes or gene expression data, has high statistical power to pinpoint the specific repair mechanisms at work. The agent-specific networks revealed roles for the histone acetyltranferase Rtt109 in the mutagenic bypass of DNA lesions and the neddylation machinery in cell-cycle regulation and genome stability, while the network induced by multiple agents implicates Irc21, an uncharacterized protein, in checkpoint control and DNA repair. Our multiconditional genetic interaction map provides a unique resource that identifies agent-specific and general DNA damage response pathways. PMID:23273983

  9. Non-coding RNAs in DNA damage response

    PubMed Central

    Liu, Yunhua; Lu, Xiongbin

    2012-01-01

    Genome-wide studies have revealed that human and other mammalian genomes are pervasively transcribed and produce thousands of regulatory non-protein-coding RNAs (ncRNAs), including miRNAs, siRNAs, piRNAs and long non-coding RNAs (lncRNAs). Emerging evidences suggest that these ncRNAs also play a pivotal role in genome integrity and stability via the regulation of DNA damage response (DDR). In this review, we discuss the recent finding on the interplay of ncRNAs with the canonical DDR signaling pathway, with a particular emphasis on miRNAs and lncRNAs. While the expression of ncRNAs is regulated in the DDR, the DDR is also subjected to regulation by those DNA damage-responsive ncRNAs. In addition, the roles of those Dicer- and Drosha-dependent small RNAs produced in the vicinity of double-strand breaks sites are also described. PMID:23226613

  10. The Yeast Copper Response Is Regulated by DNA Damage

    PubMed Central

    Dong, Kangzhen; Addinall, Stephen G.; Lydall, David

    2013-01-01

    Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast. PMID:23959798

  11. SUMO-mediated regulation of DNA damage repair and responses

    PubMed Central

    Sarangi, Prabha; Zhao, Xiaolan

    2015-01-01

    Sumoylation plays important roles during DNA damage repair and responses. Recent broad-scope and substrate-based studies have shed light on the regulation and significance of sumoylation during these processes. An emerging paradigm is that sumoylation of many DNA metabolism proteins is controlled by DNA engagement. Such “on-site modification” can explain low substrate modification levels and has important implications in sumoylation mechanisms and effects. New studies also suggest that sumoylation can regulate a process through an ensemble effect or via major substrates. Additionally, we describe new trends in the functional effects of sumoylation, such as bi-directional changes in biomolecule binding and multi-level coordination with other modifications. These emerging themes and models will stimulate our thinking and research in sumoylation and genome maintenance. PMID:25778614

  12. Hyperactivation of DNA-PK by Double-Strand Break Mimicking Molecules Disorganizes DNA Damage Response

    PubMed Central

    Quanz, Maria; Chassoux, Danielle; Berthault, Nathalie; Agrario, Céline; Sun, Jian-Sheng; Dutreix, Marie

    2009-01-01

    Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an “all-or-none” pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a “false” DNA damage signaling that disorganizes the DNA repair system. PMID:19621083

  13. DNA damage response and sphingolipid signaling in liver diseases.

    PubMed

    Nagahashi, Masayuki; Matsuda, Yasunobu; Moro, Kazuki; Tsuchida, Junko; Soma, Daiki; Hirose, Yuki; Kobayashi, Takashi; Kosugi, Shin-Ichi; Takabe, Kazuaki; Komatsu, Masaaki; Wakai, Toshifumi

    2016-09-01

    Patients with unresectable hepatocellular carcinoma (HCC) cannot generally be cured by systemic chemotherapy or radiotherapy due to their poor response to conventional therapeutic agents. The development of novel and efficient targeted therapies to increase their treatment options depends on the elucidation of the molecular mechanisms that underlie the pathogenesis of HCC. The DNA damage response (DDR) is a network of cell-signaling events that are triggered by DNA damage. Its dysregulation is thought to be one of the key mechanisms underlying the generation of HCC. Sphingosine-1-phosphate (S1P), a lipid mediator, has emerged as an important signaling molecule that has been found to be involved in many cellular functions. In the liver, the alteration of S1P signaling potentially affects the DDR pathways. In this review, we explore the role of the DDR in hepatocarcinogenesis of various etiologies, including hepatitis B and C infection and non-alcoholic steatohepatitis. Furthermore, we discuss the metabolism and functions of S1P that may affect the hepatic DDR. The elucidation of the pathogenic role of S1P may create new avenues of research into therapeutic strategies for patients with HCC. PMID:26514817

  14. Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks.

    PubMed

    Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H; Murray, Johanne M; Carr, Antony M; Elledge, Stephen J; Rhind, Nicholas

    2016-06-28

    The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes. PMID:27298342

  15. DDRprot: a database of DNA damage response-related proteins

    PubMed Central

    Andrés-León, Eduardo; Cases, Ildefonso; Arcas, Aida; Rojas, Ana M.

    2016-01-01

    The DNA Damage Response (DDR) signalling network is an essential system that protects the genome’s integrity. The DDRprot database presented here is a resource that integrates manually curated information on the human DDR network and its sub-pathways. For each particular DDR protein, we present detailed information about its function. If involved in post-translational modifications (PTMs) with each other, we depict the position of the modified residue/s in the three-dimensional structures, when resolved structures are available for the proteins. All this information is linked to the original publication from where it was obtained. Phylogenetic information is also shown, including time of emergence and conservation across 47 selected species, family trees and sequence alignments of homologues. The DDRprot database can be queried by different criteria: pathways, species, evolutionary age or involvement in (PTM). Sequence searches using hidden Markov models can be also used. Database URL: http://ddr.cbbio.es. PMID:27577567

  16. DDRprot: a database of DNA damage response-related proteins.

    PubMed

    Andrés-León, Eduardo; Cases, Ildefonso; Arcas, Aida; Rojas, Ana M

    2016-01-01

    The DNA Damage Response (DDR) signalling network is an essential system that protects the genome's integrity. The DDRprot database presented here is a resource that integrates manually curated information on the human DDR network and its sub-pathways. For each particular DDR protein, we present detailed information about its function. If involved in post-translational modifications (PTMs) with each other, we depict the position of the modified residue/s in the three-dimensional structures, when resolved structures are available for the proteins. All this information is linked to the original publication from where it was obtained. Phylogenetic information is also shown, including time of emergence and conservation across 47 selected species, family trees and sequence alignments of homologues. The DDRprot database can be queried by different criteria: pathways, species, evolutionary age or involvement in (PTM). Sequence searches using hidden Markov models can be also used.Database URL: http://ddr.cbbio.es. PMID:27577567

  17. The dynamic behavior of Ect2 in response to DNA damage

    PubMed Central

    He, Dan; Xiang, Jinnan; Li, Baojie; Liu, Huijuan

    2016-01-01

    Ect2 is a BRCT-containing guanidine exchange factor for Rho GTPases. It is essential for cytokinesis and is also involved in tumorigenesis. Since most BRCT-containing proteins are involved in DNA damage response and/or DNA repair, we tested whether Ect2 plays similar roles. We report that in primary mouse embryonic fibroblasts (MEFs), DNA damage quickly led to Ect2 relocalization to the chromatin and DNA damage foci-like structures. Ect2 knockdown did not affect foci localization of γH2AX, TopBP1, or Brca1, or activation of Atm, yet it impeded p53 Ser15 phosphorylation and activation, and resulted in defects in apoptosis and activation of S and G2/M checkpoints in response to DNA damage. These results suggest that Ect2 plays a role in DNA damage response. Interestingly, Ect2 is down-regulated at late stages of DNA damage response. Although p53 and E2F1 have been shown to regulate Ect2 transcription, DNA damage-induced Ect2 down-regulation occurred in p53−/− or Atm−/− MEFs and E2F1 knockdown cells. Instead, DNA damage-induced Ect2 down-regulation is mainly attributable to decreased protein stability. Like Ect2 knockdown, Ect2 destabilization may help the cell to recover from DNA damage response. These results suggest that Ect2 plays roles in multiple aspects of DNA damage response. PMID:27074761

  18. DNA Damage Response in Hematopoietic Stem Cell Ageing.

    PubMed

    Li, Tangliang; Zhou, Zhong-Wei; Ju, Zhenyu; Wang, Zhao-Qi

    2016-06-01

    Maintenance of tissue-specific stem cells is vital for organ homeostasis and organismal longevity. Hematopoietic stem cells (HSCs) are the most primitive cell type in the hematopoietic system. They divide asymmetrically and give rise to daughter cells with HSC identity (self-renewal) and progenitor progenies (differentiation), which further proliferate and differentiate into full hematopoietic lineages. Mammalian ageing process is accompanied with abnormalities in the HSC self-renewal and differentiation. Transcriptional changes and epigenetic modulations have been implicated as the key regulators in HSC ageing process. The DNA damage response (DDR) in the cells involves an orchestrated signaling pathway, consisting of cell cycle regulation, cell death and senescence, transcriptional regulation, as well as chromatin remodeling. Recent studies employing DNA repair-deficient mouse models indicate that DDR could intrinsically and extrinsically regulate HSC maintenance and play important roles in tissue homeostasis of the hematopoietic system. In this review, we summarize the current understanding of how the DDR determines the HSC fates and finally contributes to organismal ageing. PMID:27221660

  19. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  20. ERK3 regulates TDP2-mediated DNA damage response and chemoresistance in lung cancer cells

    PubMed Central

    Bian, Ka; Muppani, Naveen Reddy; Elkhadragy, Lobna; Wang, Wei; Zhang, Cheng; Chen, Tenghui; Jung, Sungyun; Seternes, Ole Morten; Long, Weiwen

    2016-01-01

    Posttranslational modifications (PTMs), such as phosphorylation and ubiquitination, play critical regulatory roles in the assembly of DNA damage response proteins on the DNA damage site and their activities in DNA damage repair. Tyrosyl DNA phosphodiesterase 2 (TDP2) repairs Topoisomerase 2 (Top2)-linked DNA damage, thereby protecting cancer cells against Top2 inhibitors-induced growth inhibition and cell death. The regulation of TDP2 activity by post-translational modifications in DNA repair, however, remains unclear. In the current study, we have found that ERK3, an atypical MAPK, phosphorylates TDP2 at S60 and regulates TDP2's phosphodiesterase activity, thereby cooperatively protecting lung cancer cells against Top2 inhibitors-induced DNA damage and growth inhibition. As such, our study revealed a post-translational regulation of TDP2 activity and discovered a new role of ERK3 in increasing cancer cells’ DNA damage response and chemoresistance to Top2 inhibitors. PMID:26701725

  1. Transcriptional profiling of the age-related response to genotoxic stress points to differential DNA damage response with age.

    PubMed

    Simon, Kirk; Mukundan, Anju; Dewundara, Samantha; Van Remmen, Holly; Dombkowski, Alan A; Cabelof, Diane C

    2009-09-01

    The p53 DNA damage response attenuated with age and we have evaluated downstream factors in the DNA damage response. In old animals p21 protein accumulates in the whole cell fraction but significantly declines in the nucleus, which may alter cell cycle and apoptotic programs in response to DNA damage. We evaluated the transcriptional response to DNA damage in young and old and find 2692 genes are differentially regulated in old compared to young in response to oxidative stress (p<0.005). As anticipated, the transcriptional profile of young mice is consistent with DNA damage induced cell cycle arrest while the profile of old mice is consistent with cell cycle progression in the presence of DNA damage, suggesting the potential for catastrophic accumulation of DNA damage at the replication fork. Unique sets of DNA repair genes are induced in response to damage in old and young, suggesting the types of damage accumulating differs between young and old. The DNA repair genes upregulated in old animals point to accumulation of replication-dependent DNA double strand breaks (DSB). Expression data is consistent with loss of apoptosis following DNA damage in old animals. These data suggest DNA damage responses differ greatly in young and old animals. PMID:19679149

  2. Prognostic implications of the DNA damage response pathway in glioblastoma.

    PubMed

    Seol, Ho Jun; Yoo, Hae Yong; Jin, Juyoun; Joo, Kyeung Min; Kong, Doo-Sik; Yoon, Su Jin; Yang, Heekyoung; Kang, Wonyoung; Lim, Do-Hoon; Park, Kwan; Kim, Jong Hyun; Lee, Jung-Ii; Nam, Do-Hyun

    2011-08-01

    Genomic instability and resistance to genotoxic therapies for glioblastoma (GBM) suggest aberrant DNA damage response (DDR), since DDR maintains the genomic integrity against genotoxic insults including anti-tumor therapies. To elucidate the biological and clinical meaning of DDR in GBM, we retrospectively investigated the immunohistochemical expression of DDR proteins (ATM, Chk1, Chk2, TopBP1, Rad17, p53, Nbs1, MDC1, γH2AX and RPA1) in 69 GBM surgical samples and their relation with GBM patient survival. Remarkably, higher expression of ATM revealed significantly longer overall survival (OS) and progression-free survival (PFS) (p<0.05). Upon multivariate analysis, expression level of ATM was an independent factor for longer OS (p=0.020) and longer PFS (p=0.019). Since ATM induces cell cycle arrest or apoptosis through cell cycle regulators in response to genotoxic insults, these results indicate that aberrant DDR signaling through ATM in GBM may be associated with resistance to genotoxic anti-tumor therapeutics. Conclusively, we emphasize that the identification of DDR machinery, which can be involved in unstable genomic status or genotoxic therapies in GBM, is very important to predict patient outcome. PMID:21617879

  3. Increased Sensitivity of DNA Damage Response-Deficient Cells to Stimulated Microgravity-Induced DNA Lesions

    PubMed Central

    Li, Nan; An, Lili; Hang, Haiying

    2015-01-01

    Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects. PMID:25915950

  4. DNA damage response in peripheral nervous system: coping with cancer therapy-induced DNA lesions.

    PubMed

    Englander, Ella W

    2013-08-01

    In the absence of blood brain barrier (BBB) the DNA of peripheral nervous system (PNS) neurons is exposed to a broader spectrum of endogenous and exogenous threats compared to that of the central nervous system (CNS). Hence, while CNS and PNS neurons cope with many similar challenges inherent to their high oxygen consumption and vigorous metabolism, PNS neurons are also exposed to circulating toxins and inflammatory mediators due to relative permeability of PNS blood nerve barrier (BNB). Consequently, genomes of PNS neurons incur greater damage and the question awaiting investigation is whether specialized repair mechanisms for maintenance of DNA integrity have evolved to meet the additional needs of PNS neurons. Here, I review data showing how PNS neurons manage collateral DNA damage incurred in the course of different anti-cancer treatments designed to block DNA replication in proliferating tumor cells. Importantly, while PNS neurotoxicity and concomitant chemotherapy-induced peripheral neuropathy (CIPN) are among major dose limiting barriers in achieving therapy goals, CIPN is partially reversible during post-treatment nerve recovery. Clearly, cell recovery necessitates mobilization of the DNA damage response and underscores the need for systematic investigation of the scope of DNA repair capacities in the PNS to help predict post-treatment risks to recovering neurons. PMID:23684797

  5. DNA Damage Response in Peripheral Nervous System: Coping with Cancer Therapy-Induced DNA Lesions

    PubMed Central

    Englander, Ella W

    2013-01-01

    In the absence of blood brain barrier (BBB) the DNA of peripheral nervous system (PNS) neurons is exposed to a broader spectrum of endogenous and exogenous threats compared to that of the central nervous system (CNS). Hence, while CNS and PNS neurons cope with many similar challenges inherent to their high oxygen consumption and vigorous metabolism, PNS neurons are also exposed to circulating toxins and inflammatory mediators due to relative permeability of PNS blood nerve barrier (BNB). Consequently, genomes of PNS neurons incur greater damage and the question awaiting investigation is whether specialized repair mechanisms for maintenance of DNA integrity have evolved to meet the additional needs of PNS neurons. Here, I review data showing how PNS neurons manage collateral DNA damage incurred in the course of different anti-cancer treatments designed to block DNA replication in proliferating tumor cells. Importantly, while PNS neurotoxicity and concomitant chemotherapy-induced peripheral neuropathy (CIPN) are among major dose limiting barriers in achieving therapy goals, CIPN is partially reversible during post-treatment nerve recovery. Clearly, cell recovery necessitates mobilization of the DNA damage response and underscores the need for systematic investigation of the scope of DNA repair capacities in the PNS to help predict post-treatment risks to recovering neurons. PMID:23684797

  6. Histone ubiquitylation and its roles in transcription and DNA damage response.

    PubMed

    Meas, Rithy; Mao, Peng

    2015-12-01

    DNA in human cells is constantly assaulted by endogenous and exogenous DNA damaging agents. It is vital for the cell to respond rapidly and precisely to DNA damage to maintain genome integrity and reduce the risk of mutagenesis. Sophisticated reactions occur in chromatin surrounding the damaged site leading to the activation of DNA damage response (DDR), including transcription reprogramming, cell cycle checkpoint, and DNA repair. Histone proteins around the DNA damage play essential roles in DDR, through extensive post-translational modifications (PTMs) by a variety of modifying enzymes. One PTM on histones, mono-ubiquitylation, has emerged as a key player in cellular response to DNA damage. In this review, we will (1) briefly summarize the history of histone H2A and H2B ubiquitylation (H2Aub and H2Bub, respectively), (2) discuss their roles in transcription, and (3) their functions in DDR. PMID:26422137

  7. Changing the ubiquitin landscape during viral manipulation of the DNA damage response

    PubMed Central

    Weitzman, Matthew D.; Lilley, Caroline E.; Chaurushiya, Mira S.

    2011-01-01

    Viruses often induce signaling through the same cellular cascades that are activated by damage to the cellular genome. Signaling triggered by viral proteins or exogenous DNA delivered by viruses can be beneficial or detrimental to viral infection. Viruses have therefore evolved to dissect the cellular DNA damage response pathway during infection, often marking key cellular regulators with ubiquitin to induce their degradation or change their function. Signaling controlled by ubiquitin or ubiquitin-like proteins has recently emerged as key regulator of the cellular DNA damage response. Situated at the interface between DNA damage signaling and the ubiquitin system, viruses can reveal key convergence points in this important cellular pathway. In this review, we examine how viruses harness the diversity of the cellular ubiquitin system to modulate the DNA damage signaling pathway. We discuss the implications of viral infiltration of this pathway for both the transcriptional program of the virus and for the cellular response to DNA damage. PMID:21549706

  8. DNA damage response induced by HZE particles in human cells

    NASA Astrophysics Data System (ADS)

    Chen, David; Aroumougame, Asaithamby

    Convincing evidences indicate that high-linear energy transfer (LET) ionizing radiation (IR) induced complex DNA lesions are more difficult to repair than isolated DNA lesions induced by low-LET IR; this has been associated with the increased RBE for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in high energy charged-particle irradiated human cells. We have employed an in situ method to directly monitor induction and repair of clustered DNA lesions at the single-cell level. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages determined the cellular ability to repair these damages. Importantly, examination of metaphase cells derived from HZE particle irradiated cells revealed that the extent of chromosome aberrations directly correlated with the levels of unrepaired clustered DNA lesions. In addition, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We found that complex DNA lesions induced by HZE particles were even more difficult to be repaired in organotypic 3D culture, resulting enhanced cell killing and chromosome aberrations. Our data suggest that DNA repair capability in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis. As the organotypic 3D model mimics human lung, it opens up new experimental approaches to explore the effect of radiation in vivo and will have important implications for evaluating radiation risk in human tissues.

  9. BRCA1 in the DNA damage response and at telomeres

    PubMed Central

    Rosen, Eliot M.

    2013-01-01

    Mutations of the breast and ovarian cancer susceptibility gene 1 (BRCA1) account for about 40–45% of hereditary breast cancer cases. Moreover, a significant fraction of sporadic (non-hereditary) breast and ovarian cancers exhibit reduced or absent expression of the BRCA1 protein, suggesting an additional role for BRCA1 in sporadic cancers. BRCA1 follows the classic pattern of a highly penetrant Knudsen-type tumor suppressor gene in which one allele is inactivated through a germ-line mutation and the other is mutated or deleted within the tumor. BRCA1 is a multi-functional protein but it is not fully understood which function(s) is (are) most important for tumor suppression, nor is it clear why BRCA1-mutations confer a high risk for breast and ovarian cancers and not a broad spectrum of tumor types. Here, we will review BRCA1 functions in the DNA damage response (DDR), which are likely to contribute to tumor suppression. In the process, we will highlight some of the controversies and unresolved issues in the field. We will also describe a recently identified and under-investigated role for BRCA1 in the regulation of telomeres and the implications of this role in the DDR and cancer suppression. PMID:23802008

  10. Expression of DNA damage response genes indicate progressive breast tumors.

    PubMed

    Gochhait, Sailesh; Dar, Surabhi; Pal, Ranjana; Gupta, Pawan; Bamezai, Rameshwar N K

    2009-01-18

    To assess how the abnormal expression of DNA damage response (DDR) genes correlate with oncogenesis, we analyzed mRNA levels of ATM-CHK2-P53 axis in 65 sporadic breast tumors by real-time PCR followed by evaluation of P53 protein and its activation status in representative samples. Univariate analysis showed a significantly higher transcript level for ATM (P=0.002), MDM2 (P=0.015) and p21 (P=0.013) in stage 1 tumors when compared against those of later stages. Although p53 transcript levels showed the characteristic increase in stage 1, a fourfold increase of p53 in N3 tumors than other nodal stages (P=0.0007) significantly increased its expression in stage 3B. The accumulated p53 at stage 3B, confirmed also at the protein level (P=0.012), was rendered nonfunctional by reduced P53 activation (p-P53Ser15; P=0.00007) or increased rate of mutation, substantiated further by the corresponding failure of upregulation of downstream genes, MDM2 and p21. We conclude that the alteration of DDR expression facilitates tumor progression and its possible therapeutic implications need to be studied in future. PMID:18805634

  11. Sumoylation of MDC1 is important for proper DNA damage response.

    PubMed

    Luo, Kuntian; Zhang, Haoxing; Wang, Liewei; Yuan, Jian; Lou, Zhenkun

    2012-06-29

    In response to DNA damage, many DNA damage factors, such as MDC1 and 53BP1, redistribute to sites of DNA damage. The mechanism governing the turnover of these factors at DNA damage sites, however, remains enigmatic. Here, we show that MDC1 is sumoylated following DNA damage, and the sumoylation of MDC1 at Lys1840 is required for MDC1 degradation and removal of MDC1 and 53BP1 from sites of DNA damage. Sumoylated MDC1 is recognized and ubiquitinated by the SUMO-targeted E3 ubiquitin ligase RNF4. Mutation of the MDC1 Lys 1840 (K1840R) results in impaired CtIP, replication protein A, and Rad51 accumulation at sites of DNA damage and defective homologous recombination (HR). The HR defect caused by MDC1K1840R mutation could be rescued by 53BP1 downregulation. These results reveal the intricate dynamics governing the assembly and disassembly of DNA damage factors at sites of DNA damage for prompt response to DNA damage. PMID:22635276

  12. p19(Arf) is required for the cellular response to chronic DNA damage.

    PubMed

    Bieging-Rolett, K T; Johnson, T M; Brady, C A; Beaudry, V G; Pathak, N; Han, S; Attardi, L D

    2016-08-18

    The p53 tumor suppressor is a stress sensor, driving cell cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19(Arf) tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19(Arf)-independent. Accordingly, p19(Arf)-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19(Arf) is required for replicative senescence, a condition associated with an activated DNA damage response, as p19(Arf)-/- MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19(Arf) is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19(Arf) has a specific role. We therefore examined the role of p19(Arf) in cellular responses to chronic, low-dose DNA-damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5 G y γ-irradiation daily or 150 μM hydroxyurea, a replication stress inducer. In contrast to their response to acute DNA damage, p19(Arf)-/- MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19(Arf) in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19(Arf)-/- MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19(Arf) in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA-damaging agent treatment is insufficient to promote senescence in p19(Arf)-/- MEFs, suggesting that the role of p19(Arf) in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19(Arf) for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19(Arf) serves as a tumor suppressor. Moreover, our study helps reconcile reports

  13. p19Arf is required for the cellular response to chronic DNA damage

    PubMed Central

    Bieging-Rolett, Kathryn T.; Johnson, Thomas M.; Brady, Colleen A.; Beaudry, Veronica G.; Pathak, Navneeta; Han, Shuo; Attardi, Laura D.

    2015-01-01

    The p53 tumor suppressor is a stress sensor, driving cell-cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19Arf tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19Arf-independent. Accordingly, p19Arf-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19Arf is required for replicative senescence, a condition associated with an activated DNA damage response, as p19Arf−/− MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19Arf is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19Arf has a specific role. We therefore examined the role of p19Arf in cellular responses to chronic, low-dose DNA damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5 Gy γ-irradiation daily or 150μM hydroxyurea, a replication stress-inducer. In contrast to their response to acute DNA damage, p19Arf−/− MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19Arf in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19Arf−/− MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19Arf in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA damaging agent treatment is insufficient to promote senescence in p19Arf−/− MEFs, suggesting that the role of p19Arf in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19Arf for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19Arf serves as a tumor suppressor. Moreover, our study helps reconcile reports suggesting crucial

  14. Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response

    PubMed Central

    Moreli, Jusciele B.; Santos, Janine H.; Lorenzon-Ojea, Aline Rodrigues; Corrêa-Silva, Simone; Fortunato, Rodrigo S.; Rocha, Clarissa Ribeiro; Rudge, Marilza V.; Damasceno, Débora C.; Bevilacqua, Estela; Calderon, Iracema M.

    2016-01-01

    Objective: Investigate the DNA damage and its cellular response in blood samples from both mother and the umbilical cord of pregnancies complicated by hyperglycemia. Methods: A total of 144 subjects were divided into 4 groups: normoglycemia (ND; 46 cases), mild gestational hyperglycemia (MGH; 30 cases), gestational diabetes mellitus (GDM; 45 cases) and type-2 diabetes mellitus (DM2; 23 cases). Peripheral blood mononuclear cell (PBMC) isolation and/or leukocytes from whole maternal and umbilical cord blood were obtained from all groups at delivery. Nuclear and mitochondrial DNA damage were measured by gene-specific quantitative PCR, and the expression of mRNA and proteins involved in the base excision repair (BER) pathway were assessed by real-time qPCR and Western blot, respectively. Apoptosis was measured in vitro experiments by caspase 3/7 activity and ATP levels. Results: GDM and DM2 groups were characterized by an increase in oxidative stress biomarkers, an increase in nuclear and mitochondrial DNA damage, and decreased expression of mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1) involved in BER. The levels of hyperglycemia were associated with the in vitro apoptosis pathway. Blood levels of DNA damage in umbilical cord were similar among the groups. Newborns of diabetic mothers had increased expression of BER mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1, POLβ and FEN1). A diabetes-like environment was unable to induce apoptosis in the umbilical cord blood cells. Conclusions: Our data show relevant asymmetry between maternal and fetal blood cell susceptibility to DNA damage and apoptosis induction. Maternal cells seem to be more predisposed to changes in an adverse glucose environment. This may be due to differential ability in upregulating multiple genes involved in the activation of DNA repair response, especially the BER mechanism. However if this study shows a more effective adaptive response by the fetal organism, it also calls for

  15. DNA Damage Responses in Prokaryotes: Regulating Gene Expression, Modulating Growth Patterns, and Manipulating Replication Forks

    PubMed Central

    Kreuzer, Kenneth N.

    2013-01-01

    Recent advances in the area of bacterial DNA damage responses are reviewed here. The SOS pathway is still the major paradigm of bacterial DNA damage response, and recent studies have clarified the mechanisms of SOS induction and key physiological roles of SOS including a very major role in genetic exchange and variation. When considering diverse bacteria, it is clear that SOS is not a uniform pathway with one purpose, but rather a platform that has evolved for differing functions in different bacteria. Relating in part to the SOS response, the field has uncovered multiple apparent cell-cycle checkpoints that assist cell survival after DNA damage and remarkable pathways that induce programmed cell death in bacteria. Bacterial DNA damage responses are also much broader than SOS, and several important examples of LexA-independent regulation will be reviewed. Finally, some recent advances that relate to the replication and repair of damaged DNA will be summarized. PMID:24097899

  16. Ciliogenesis and the DNA damage response: a stressful relationship.

    PubMed

    Johnson, Colin A; Collis, Spencer J

    2016-01-01

    Both inherited and sporadic mutations can give rise to a plethora of human diseases. Through myriad diverse cellular processes, sporadic mutations can arise through a failure to accurately replicate the genetic code or by inaccurate separation of duplicated chromosomes into daughter cells. The human genome has therefore evolved to encode a large number of proteins that work together with regulators of the cell cycle to ensure that it remains error-free. This is collectively known as the DNA damage response (DDR), and genome stability mechanisms involve a complex network of signalling and processing factors that ensure redundancy and adaptability of these systems. The importance of genome stability mechanisms is best illustrated by the dramatic increased risk of cancer in individuals with underlying disruption to genome maintenance mechanisms. Cilia are microtubule-based sensory organelles present on most vertebrate cells, where they facilitate transduction of external signals into the cell. When not embedded within the specialised ciliary membrane, components of the primary cilium's basal body help form the microtubule organising centre that controls cellular trafficking and the mitotic segregation of chromosomes. Ciliopathies are a collection of diseases associated with functional disruption to cilia function through a variety of different mechanisms. Ciliopathy phenotypes can vary widely, and although some cellular overgrowth phenotypes are prevalent in a subset of ciliopathies, an increased risk of cancer is not noted as a clinical feature. However, recent studies have identified surprising genetic and functional links between cilia-associated proteins and genome maintenance factors. The purpose of this mini-review is to therefore highlight some of these discoveries and discuss their implications with regards to functional crosstalk between the DDR and ciliogenesis pathways, and how this may impact on the development of human disease. PMID:27335639

  17. The DNA Damage Response: Making it safe to play with knives

    PubMed Central

    Ciccia, Alberto; Elledge, Stephen J.

    2010-01-01

    Damage to our genetic material is an ongoing threat to both our ability to faithfully transmit genetic information to our offspring as well as our own survival. To respond to these threats, eukaryotes have evolved the DNA Damage Response (DDR). The DDR is a complex signal transduction pathway that has the ability to sense DNA damage and transduce this information to the cell to influence cellular responses to DNA damage. Cells possess an arsenal of enzymatic tools capable of remodeling and repairing DNA, however, their activities must be tightly regulated in a temporal, spatial and DNA lesion-appropriate fashion to optimize repair and prevent unnecessary and potentially deleterious alterations in the structure of DNA during normal cellular processes. This review will focus on how the DDR controls DNA repair and the phenotypic consequences of defects in these critical regulatory functions in mammals. PMID:20965415

  18. The activation of DNA damage detection and repair responses in cleavage-stage rat embryos by a damaged paternal genome.

    PubMed

    Grenier, Lisanne; Robaire, Bernard; Hales, Barbara F

    2012-06-01

    Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence. PMID:22454429

  19. Stress responses to DNA damaging agents in the human colon carcinoma cell line, RKO.

    PubMed

    Beard, S E; Capaldi, S R; Gee, P

    1996-11-01

    DNA damage results from a wide variety of external agents such as chemicals and radiation. The consequences of exposure to agents that damage DNA have been traditionally studied from the perspective of cell survival and mutagenesis. Mutations are late endpoints of DNA damage. Cells respond to the earlier stages of DNA damage by inducing the expression of several genes, including those specific of the nature of the lesion. These early transcriptional responses are likely to predetermine the later fate of the damaged cell. Genes activated during this early response include those involved in DNA repair, replication, and growth control. We are interested in the transcriptional mechanisms by which cells respond to DNA damaging agents. To facilitate the measurement of gene induction, we used seven different reporter constructs integrated stably into the RKO cell line derived from a human colon carcinoma. These constructs were derived from promoters and/or response elements isolated from genes associated with DNA damage responses in human cells, and were fused to the bacterial reporter gene, choramphenicol acetyl transferase (CAT). The cell lines generated in this manner contain the promoters and/or response elements representing DNA polymerase beta, p53, gadd (growth arrest and DNA damage) 45 and 153, c-fos, TPA response element, and tissue-type plasminogen activator. These recombinant cell lines were assembled in a 96-well microtiter plate permitting their simultaneous exposure to compounds and subsequent CAT protein measurement. This assembly has been designated the CAT-Tox (D) assay. These cell lines were exposed to different classes of DNA damaging agents including those which covalently join bases to form dimers (e.g., UVC irradiation), generate DNA adducts by alkylation (e.g., methylmethane sulfonate [MMS], ethylmethane sulfonate [EMS], N-methyl-N-nitro-N-nitrosoguanine [MNNG], dimethylnitrosamine [DMN]), cross-link DNA (e.g., mitomycin C), and inhibit DNA

  20. Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.

    PubMed

    Wingert, Susanne; Rieger, Michael A

    2016-07-01

    Hematopoietic stem cells (HSCs) sustain lifelong blood cell regeneration by balancing their ability for self-renewal with their ability to differentiate into all blood cell types. To prevent organ exhaustion and malignant transformation, long-lived HSCs, in particular, must be protected from exogenous and endogenous stress, which cause severe DNA damage. When DNA is damaged, distinct DNA repair mechanisms and cell fate controls occur in adult HSCs compared with committed cells. Growth arrest and DNA damage-inducible 45 alpha (GADD45A) is known to coordinate a variety of cellular stress responses, indicating the molecule is an important stress mediator. So far, the function of GADD45A in hematopoietic stem and progenitor cells is controversial and appears highly dependent on the cell type and stress stimulus. Recent studies have analyzed its role in cell fate decision control of prospectively isolated HSCs and have revealed unexpected functions of GADD45A, as discussed here. The upregulation of GADD45A by DNA damage-causing conditions results in enhanced HSC differentiation, probably to efficiently eliminate aberrant HSCs from the system. These findings, in concert with a few studies on other stem cell systems, have led us to propose DNA damage-induced differentiation as a novel DNA damage response mechanism in stem cells that circumvents the fatal consequences of cumulative DNA damage in the stem cell compartment. PMID:27262218

  1. The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response.

    PubMed

    Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun

    2015-12-15

    Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response. PMID:26400172

  2. The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response

    PubMed Central

    Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun

    2015-01-01

    Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response. PMID:26400172

  3. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    SciTech Connect

    Ganesan, Shanthi Keating, Aileen F.

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  4. Alkaline ceramidase 2 and its bioactive product sphingosine are novel regulators of the DNA damage response

    PubMed Central

    Xu, Ruijuan; Wang, Kai; Mileva, Izolda; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

    2016-01-01

    Human cells respond to DNA damage by elevating sphingosine, a bioactive sphingolipid that induces programmed cell death (PCD) in response to various forms of stress, but its regulation and role in the DNA damage response remain obscure. Herein we demonstrate that DNA damage increases sphingosine levels in tumor cells by upregulating alkaline ceramidase 2 (ACER2) and that the upregulation of the ACER2/sphingosine pathway induces PCD in response to DNA damage by increasing the production of reactive oxygen species (ROS). Treatment with the DNA damaging agent doxorubicin increased both ACER2 expression and sphingosine levels in HCT116 cells in a dose-dependent manner. ACER2 overexpression increased sphingosine in HeLa cells whereas knocking down ACER2 inhibited the doxorubicin-induced increase in sphingosine in HCT116 cells, suggesting that DNA damage elevates sphingosine by upregulating ACER2. Knocking down ACER2 inhibited an increase in the apoptotic and necrotic cell population and the cleavage of poly ADP ribose polymerase (PARP) in HCT116 cells in response to doxorubicin as well as doxorubicin-induced release of lactate dehydrogenase (LDH) from these cells. Similar to treatment with doxorubicin, ACER2 overexpression induced an increase in the apoptotic and necrotic cell population and PARP cleavage in HeLa cells and LDH release from cells, suggesting that ACER2 upregulation mediates PCD in response to DNA damage through sphingosine. Mechanistic studies demonstrated that the upregulation of the ACER2/sphingosine pathway induces PCD by increasing ROS levels. Taken together, these results suggest that the ACER2/sphingosine pathway mediates PCD in response to DNA damage through ROS production. PMID:26943039

  5. Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2)

    PubMed Central

    Pérez-Caro, M; Bermejo-Rodríguez, C; González-Herrero, I; Sánchez-Beato, M; Piris, M A; Sánchez-García, I

    2008-01-01

    Snai2-deficient cells are radiosensitive to DNA damage. The function of Snai2 in response to DNA damage seems to be critical for its function in normal development and cancer. Here, we applied a functional genomics approach that combined gene-expression profiling and computational molecular network analysis to obtain global dissection of the Snai2-dependent transcriptional response to DNA damage in primary mouse embryonic fibroblasts (MEFs), which undergo p53-dependent growth arrest in response to DNA damage. Although examination of the response showed that overall expression of p53 target gene expression patterns was similarly altered in both control and Snai2-deficient cells, we have identified and validated candidate Snai2 target genes linked to Snai2 gene function in response to DNA damage. This work defines for the first time the effect of Snai2 on p53 target genes in cells undergoing growth arrest, elucidates the Snai2-dependent molecular network induced by DNA damage, points to novel putative Snai2 targets, and suggest a mechanistic model, which has implications for cancer management. PMID:18182996

  6. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  7. DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy.

    PubMed

    Fayzullina, Saniya; Martin, Lee J

    2016-09-01

    We studied DNA damage response (DDR) and DNA repair capacities of skeletal muscle cells from a mouse model of infantile spinal muscular atrophy (SMA) caused by loss-of-function mutation of survival of motor neuron (Smn). Primary myocyte cultures derived from skeletal muscle satellite cells of neonatal control and mutant SMN mice had similar myotube length, myonuclei, satellite cell marker Pax7 and differentiated myotube marker myosin, and acetylcholine receptor clustering. DNA damage was induced in differentiated skeletal myotubes by γ-irradiation, etoposide, and methyl methanesulfonate (MMS). Unexposed control and SMA myotubes had stable genome integrity. After γ-irradiation and etoposide, myotubes repaired most DNA damage equally. Control and mutant myotubes exposed to MMS exhibited equivalent DNA damage without repair. Control and SMA myotube nuclei contained DDR proteins phospho-p53 and phospho-H2AX foci that, with DNA damage, dispersed and then re-formed similarly after recovery. We conclude that mouse primary satellite cell-derived myotubes effectively respond to and repair DNA strand-breaks, while DNA alkylation repair is underrepresented. Morphological differentiation, genome stability, genome sensor, and DNA strand-break repair potential are preserved in mouse SMA myocytes; thus, reduced SMN does not interfere with myocyte differentiation, genome integrity, and DNA repair, and faulty DNA repair is unlikely pathogenic in SMA. PMID:27452406

  8. The cellular response to DNA damage: A focus on MDC1 and its interacting proteins

    PubMed Central

    Coster, Gideon

    2010-01-01

    The DNA damage response (DDR) is comprised of a network of proteins that respond to DNA damage. Mediator of DNA Damage Checkpoint 1 (MDC1) plays an early and important role in the DDR. Recent data show that MDC1 binds multiple proteins that participate in various aspects of the DDR, positioning it at the core of the DDR. Furthermore, interactions with non-DDR proteins were also revealed, suggesting novel roles for MDC1. In this review we provide a comprehensive overview of all known MDC1-binding proteins and discuss their role. We present these binding partners according to their function, thereby providing the reader with a detailed and updated overview of the cellular response to DNA damage. We discuss more recent findings in detail and conclude by presenting the challenges the field faces in the future. PMID:21326949

  9. PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage.

    PubMed

    Gresko, E; Ritterhoff, S; Sevilla-Perez, J; Roscic, A; Fröbius, K; Kotevic, I; Vichalkovski, A; Hess, D; Hemmings, B A; Schmitz, M L

    2009-02-01

    The promyelocytic leukemia (PML) tumor suppressor protein, a central regulator of cell proliferation and apoptosis, is frequently fused to the retinoic acid receptor-alpha (RARalpha) in acute PML. Here we show the interaction of PML with another tumor suppressor protein, the serine/threonine kinase homeodomain-interacting protein kinase (HIPK2). In response to DNA damage, HIPK2 phosphorylates PML at serines 8 and 38. Although HIPK2-mediated phosphorylation of PML occurs early during the DNA damage response, the oncogenic PML-RARalpha fusion protein is phosphorylated with significantly delayed kinetics. DNA damage or HIPK2 expression leads to the stabilization of PML and PML-RARalpha proteins. The N-terminal phosphorylation sites contribute to the DNA damage-induced PML SUMOylation and are required for the ability of PML to cooperate with HIPK2 for the induction of cell death. PMID:19015637

  10. LEM-3 - A LEM domain containing nuclease involved in the DNA damage response in C. elegans.

    PubMed

    Dittrich, Christina M; Kratz, Katja; Sendoel, Ataman; Gruenbaum, Yosef; Jiricny, Josef; Hengartner, Michael O

    2012-01-01

    The small nematode Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage - possibly by promoting the reorganization of damaged chromatin. PMID:22383942

  11. Centrosomes in the DNA damage response--the hub outside the centre.

    PubMed

    Mullee, Lisa I; Morrison, Ciaran G

    2016-01-01

    Here, we review how DNA damage affects the centrosome and how centrosomes communicate with the DNA damage response (DDR) apparatus. We discuss how several proteins of the DDR are found at centrosomes, including the ATM, ATR, CHK1 and CHK2 kinases, the BRCA1 ubiquitin ligase complex and several members of the poly(ADP-ribose) polymerase family. Stereotypical centrosome organisation, in which two centriole barrels are orthogonally arranged in a roughly toroidal pericentriolar material (PCM), is strongly affected by exposure to DNA-damaging agents. We describe the genetic dependencies and mechanisms for how the centrioles lose their close association, and the PCM both expands and distorts after DNA damage. Another consequence of genotoxic stress is that centrosomes undergo duplication outside the normal cell cycle stage, meaning that centrosome amplification is commonly seen after DNA damage. We discuss several potential mechanisms for how centrosome numbers become dysregulated after DNA damage and explore the links between the DDR and the PLK1- and separase-dependent mechanisms that drive centriole separation and reduplication. We also describe how centrosome components, such as centrin2, are directly involved in responding to DNA damage. This review outlines current questions on the involvement of centrosomes in the DDR. PMID:26614090

  12. Perspectives on the DNA damage and replication checkpoint responses in Saccharomyces cerevisiae

    PubMed Central

    Putnam, Christopher D.; Jaehnig, Eric J.; Kolodner, Richard D.

    2009-01-01

    The DNA damage and replication checkpoints are believed to primarily slow the progression of the cell cycle to allow DNA repair to occur. Here we summarize known aspects of the Saccharomyces cerevisiae checkpoints including how these responses are integrated into downstream effects on the cell cycle, chromatin, DNA repair, and cytoplasmic targets. Analysis of the transcriptional response demonstrates that it is far more complex and less relevant to the repair of DNA damage than the bacterial SOS response. We also address more speculative questions regarding potential roles of the checkpoint during the normal S-phase and how current evidence hints at a checkpoint activation mechanism mediated by positive feedback that amplifies initial damage signals above a minimium threshold. PMID:19477695

  13. Activation of a DNA damage checkpoint response in a TAF1-defective cell line.

    PubMed

    Buchmann, Ann M; Skaar, Jeffrey R; DeCaprio, James A

    2004-06-01

    Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response. PMID:15169897

  14. Activation of a DNA Damage Checkpoint Response in a TAF1-Defective Cell Line

    PubMed Central

    Buchmann, Ann M.; Skaar, Jeffrey R.; DeCaprio, James A.

    2004-01-01

    Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G1 phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G1. These results suggest that a defect in TAF1 can elicit a DNA damage response. PMID:15169897

  15. The DNA damage-induced cell death response: a roadmap to kill cancer cells.

    PubMed

    Matt, Sonja; Hofmann, Thomas G

    2016-08-01

    Upon massive DNA damage cells fail to undergo productive DNA repair and trigger the cell death response. Resistance to cell death is linked to cellular transformation and carcinogenesis as well as radio- and chemoresistance, making the underlying signaling pathways a promising target for therapeutic intervention. Diverse DNA damage-induced cell death pathways are operative in mammalian cells and finally culminate in the induction of programmed cell death via activation of apoptosis or necroptosis. These signaling routes affect nuclear, mitochondria- and plasma membrane-associated key molecules to activate the apoptotic or necroptotic response. In this review, we highlight the main signaling pathways, molecular players and mechanisms guiding the DNA damage-induced cell death response. PMID:26791483

  16. MicroRNA-138 modulates DNA damage response by repressing histone H2AX expression

    PubMed Central

    Wang, Yemin; Huang, Jen-Wei; Li, Ming; Cavenee, Webster K.; Mitchell, Patrick S.; Zhou, Xiaofeng; Tewari, Muneesh; Furnari, Frank B.; Taniguchi, Toshiyasu

    2011-01-01

    Precise regulation of DNA damage response is crucial for cellular survival after DNA damage, and its abrogation often results in genomic instability in cancer. Phosphorylated histone H2AX (γH2AX) forms nuclear foci at sites of DNA damage and facilitates DNA damage response and repair. MicroRNAs are short, non-protein-encoding RNA molecules, which post-transcriptionally regulate gene expression by repressing translation of and/or degrading mRNA. How microRNAs modulate DNA damage response is largely unknown. In this study, we developed a cell-based screening assay utilizing ionizing radiation-induced γH2AX foci formation in a human osteosarcoma cell line, U2OS, as the readout. By screening a library of human microRNA mimics, we identified several microRNAs that inhibited γH2AX foci formation. Among them, miR-138 directly targeted the histone H2AX 3′-UTR, reduced histone H2AX expression and induced chromosomal instability after DNA damage. Overexpression of miR-138 inhibited homologous recombination and enhanced cellular sensitivity to multiple DNA damaging agents (cisplatin, camptothecin, and ionizing radiation). Reintroduction of histone H2AX in miR-138 overexpressing cells attenuated miR-138-mediated sensitization to cisplatin and camptothecin. Our study suggests that miR-138 is an important regulator of genomic stability and a potential therapeutic agent to improve the efficacy of radiotherapy and chemotherapy with DNA damaging agents. PMID:21693595

  17. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, affects on the cellular response to DNA damage induced by exposures to radiation or other toxic chemicals will have an impact on the radiation risks for the astronauts, as well as on the mutation rate in microorganisms, is still an open question. Although the possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on the cellular response to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induces DNA damages including the double strand breaks (DSB) similar to the ionizing radiation. Damage in the DNA was measured by the phosphorylation of a histone protein H2AX (-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in the DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ti-67 signals. Our results suggested that the difference in -H2AX between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect the response of the DNA damage response genes to bleomycin treatment.

  18. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  19. DNA damage responsive microRNAs misexpressed in human cancer modulate therapy sensitivity

    PubMed Central

    van Jaarsveld, Marijn T.M.; Wouters, Maikel D.; Boersma, Antonius W.M.; Smid, Marcel; van IJcken, Wilfred F.J.; Mathijssen, Ron H.J.; Hoeijmakers, Jan H.J.; Martens, John W. M.; van Laere, Steven; Wiemer, Erik A.C.; Pothof, Joris

    2015-01-01

    The DNA damage response (DDR) is activated upon DNA damage and prevents accumulation of mutations and chromosomal rearrangements, both driving carcinogenesis. Tumor cells often have defects in the DDR, which in combination with continuous cell proliferation are exploited by genotoxic cancer therapies. Most cancers, overcome initial sensitivity and develop drug resistance, e.g. by modulation of the DDR. Not much is known, however, about DNA damage responsive microRNAs in cancer therapy resistance. Therefore, we mapped temporal microRNA expression changes in primary breast epithelial cells upon low and high dose exposure to the DNA damaging agents ionizing radiation and cisplatin. A third of all DDR microRNAs commonly regulated across all treatments was also misexpressed in breast cancer, indicating a DDR defect. We repeated this approach in primary lung epithelial cells and non-small cell lung cancer samples and found that more than 40% of all DDR microRNAs was deregulated in non-small cell lung cancer. Strikingly, the microRNA response upon genotoxic stress in primary breast and lung epithelial cells was markedly different, although the biological outcome of DNA damage signaling (cell death/senescence or survival) was similar. Several DDR microRNAs deregulated in cancer modulated sensitivity to anti-cancer agents. In addition we were able to distinguish between microRNAs that induced resistance by potentially inducing quiescence (miR-296-5p and miR-382) or enhancing DNA repair or increased DNA damage tolerance (miR-21). In conclusion, we provide evidence that DNA damage responsive microRNAs are frequently misexpressed in human cancer and can modulate chemotherapy sensitivity. PMID:24462518

  20. Arsenic Disruption of DNA Damage Responses-Potential Role in Carcinogenesis and Chemotherapy.

    PubMed

    Muenyi, Clarisse S; Ljungman, Mats; States, J Christopher

    2015-01-01

    Arsenic is a Class I human carcinogen and is widespread in the environment. Chronic arsenic exposure causes cancer in skin, lung and bladder, as well as in other organs. Paradoxically, arsenic also is a potent chemotherapeutic against acute promyelocytic leukemia and can potentiate the cytotoxic effects of DNA damaging chemotherapeutics, such as cisplatin, in vitro. Arsenic has long been implicated in DNA repair inhibition, cell cycle disruption, and ubiquitination dysregulation, all negatively impacting the DNA damage response and potentially contributing to both the carcinogenic and chemotherapeutic potential of arsenic. Recent studies have provided mechanistic insights into how arsenic interferes with these processes including disruption of zinc fingers and suppression of gene expression. This review discusses these effects of arsenic with a view toward understanding the impact on the DNA damage response. PMID:26404387

  1. Evaluation of cytotoxicity and DNA damage response with analysis of intracellular ATM signaling pathways.

    PubMed

    Bandi, Sriram; Viswanathan, Preeti; Gupta, Sanjeev

    2014-06-01

    Maintenance of genome integrity by preventing and overcoming DNA damage is critical for cell survival. Deficiency or aberrancy in the DNA damage response, for example, through ataxia telangiectasia mutated (ATM) signaling, lead to pathophysiological perturbations in organs throughout the body. Therefore, control of DNA damage is of major interest for development of therapeutic agents. Such efforts will greatly benefit from convenient and simple diagnostic and/or drug development tools to demonstrate whether ATM and related genes have been activated and to then determine whether these have been returned to normal levels of activity because pathway members sense and also repair DNA damage. To overcome difficulties in analyzing differences in multitudinous ATM pathway members following DNA damage, we measured ATM promoter activity with a fluorescent td-Tomato reporter gene to interrogate the global effects of ATM signaling pathways. In cultured HuH-7 cell line derived from human hepatocellular carcinoma, cis-platinum, acetaminophen, or hydrogen peroxide caused DNA strand breaks and ATM pathway activation as shown by γH2AX expression, which in turn, led to rapid and sustained increases in ATM promoter activity. This assay of ATM promoter activity identified biological agents capable of controlling cellular DNA damage in toxin-treated HuH-7 cells and in mice after onset of drug-induced acute liver failure. Therefore, the proposed assay of ATM promoter activity in HuH-7 cells was appropriately informative for treating DNA damage. High-throughput screens using ATM promoter activation will be helpful for therapeutic development in DNA damage-associated abnormal ATM signaling in various cell types and organs. PMID:24927134

  2. DDRI-9: a novel DNA damage response inhibitor that blocks mitotic progression

    PubMed Central

    Kim, Yun-Hee; Kim, Kyung-Tae; Kim, Sunshin; Lee, Chang-Hun

    2016-01-01

    The DNA damage response (DDR) is an emerging target for cancer therapy. By modulating the DDR, including DNA repair and cell cycle arrest, the efficacy of anticancer drugs can be enhanced and side effects reduced. We previously screened a chemical library and identified novel DDR inhibitors including DNA damage response inhibitor-9 (DDRI-9; 1H-Purine-2,6-dione,7-[(4-fluorophenyl)methyl]-3,7-dihydro-3-methyl-8-nitro). In this study, we characterized DDRI-9 activity and found that it inhibited phosphorylated histone variant H2AX foci formation upon DNA damage, delayed DNA repair, and enhanced the cytotoxicity of etoposide and ionizing radiation. It also reduced the foci formation of DNA repair-related proteins, including the protein kinase ataxia-telangiectasia mutated, DNA-dependent protein kinase, breast cancer type 1 susceptibility protein, and p53-binding protein 1, but excluding mediator of DNA damage checkpoint protein 1. Cell cycle analysis revealed that DDRI-9 blocked mitotic progression. Like other mitotic inhibitors, DDRI-9 treatment resulted in the accumulation of mitotic protein and induced cell death. Thus, DDRI-9 may affect both DDR signal amplification and mitotic progression. This study suggests that DDRI-9 is a good lead molecule for the development of anticancer drugs. PMID:26848527

  3. Smoking-promoted oxidative DNA damage response is highly correlated to lung carcinogenesis

    PubMed Central

    Li, Miao; Zhou, Hongbin; Lv, Dan; Deng, Zaichun; Ying, Songmin; Chen, Zhihua; Li, Wen; Shen, Huahao

    2016-01-01

    Oxidative stress induced by tobacco smoking is one of the main causes of DNA damage and is known to be involved in various cancers. Smoking is the leading cause of lung cancer, while the role of cigarette smoke-induced oxidative DNA damage response during lung carcinogenesis is largely unknown. In this study, we investigated oxidative DNA damage response levels in smoking and nonsmoking patients with lung cancer, and evaluated the potential diagnostic value of 8-OHdG for lung cancer. We observed a higher level of 8-OHdG expression and secretion in airways of lung cancer patients than that of noncancer controls. 8-OHdG expression was associated with the TNM stages. Additionally, cigarette smoke-induced oxidative DNA damage response was observed in bronchial epithelial cells in vitro and in vivo. A statistical significance correlation was found between the levels of 8-OHdG and smoking index. With a cut-off value of 2.86 ng/ml, 8-OHdG showed a sensitivity and specificity of 70.0% and 73.7%, respectively, to identify a patient with lung cancer. These findings not only underscore the importance of smoking in oxidative DNA damage response of lung cancer patients, but also suggest 8-OHdG as a potential diagnostic biomarker for lung cancer. PMID:26942876

  4. Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

    PubMed

    Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

    2005-04-15

    Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage. PMID:15837426

  5. Differential response to DNA damage may explain different cancer susceptibility between small and large intestine.

    PubMed

    Hong, Mee Young; Turner, Nancy D; Carroll, Raymond J; Chapkin, Robert S; Lupton, Joanne R

    2005-07-01

    Although large intestine (LI) cancer is the second-leading cause of cancer-related deaths in the United States, small intestine (SI) cancer is relatively rare. Because oxidative DNA damage is one possible initiator of tumorigenesis, we investigated if the SI is protected against cancer because of a more appropriate response to oxidative DNA damage compared with the LI. Sixty rats were allocated to three treatment groups: 3% dextran sodium sulfate (DSS, a DNA-oxidizing agent) for 48 hrs, withdrawal (DSS for 48 hrs + DSS withdrawal for 48 hrs), or control (no DSS). The SI, compared with the LI, showed greater oxidative DNA damage (P < 0.001) as determined using a quantitative immunohistochemical analysis of 8-oxodeoxyguanosine (8-oxodG). The response to the DNA adducts in the SI was greater than in the LI. The increase of TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive apoptosis after DSS treatment was greater in the SI compared with the LI (P < 0.001), and there was a positive correlation (P = 0.031) between DNA damage and apoptosis in the SI. Morphologically, DSS caused an extensive loss of crypt structure shown in lower crypt height (P = 0.006) and the number of intact crypts (P = 0.0001) in the LI, but not in the SI. These data suggest that the SI may be more protected against cancer by having a more dynamic response to oxidative damage that maintains crypt morphology, whereas the response of the LI makes it more susceptible to loss of crypt architecture. These differential responses to oxidative DNA damage may contribute to the difference in cancer susceptibility between these two anatomic sites of the intestine. PMID:15985621

  6. Potential Relationship between Inadequate Response to DNA Damage and Development of Myelodysplastic Syndrome

    PubMed Central

    Zhou, Ting; Chen, Peishuai; Gu, Jian; Bishop, Alexander J. R.; Scott, Linda M.; Hasty, Paul; Rebel, Vivienne I.

    2015-01-01

    Hematopoietic stem cells (HSCs) are responsible for the continuous regeneration of all types of blood cells, including themselves. To ensure the functional and genomic integrity of blood tissue, a network of regulatory pathways tightly controls the proliferative status of HSCs. Nevertheless, normal HSC aging is associated with a noticeable decline in regenerative potential and possible changes in other functions. Myelodysplastic syndrome (MDS) is an age-associated hematopoietic malignancy, characterized by abnormal blood cell maturation and a high propensity for leukemic transformation. It is furthermore thought to originate in a HSC and to be associated with the accrual of multiple genetic and epigenetic aberrations. This raises the question whether MDS is, in part, related to an inability to adequately cope with DNA damage. Here we discuss the various components of the cellular response to DNA damage. For each component, we evaluate related studies that may shed light on a potential relationship between MDS development and aberrant DNA damage response/repair. PMID:25569081

  7. DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies

    PubMed Central

    Wang, Qi-En

    2015-01-01

    The identification of cancer stem cells (CSCs) that are responsible for tumor initiation, growth, metastasis, and therapeutic resistance might lead to a new thinking on cancer treatments. Similar to stem cells, CSCs also display high resistance to radiotherapy and chemotherapy with genotoxic agents. Thus, conventional therapy may shrink the tumor volume but cannot eliminate cancer. Eradiation of CSCs represents a novel therapeutic strategy. CSCs possess a highly efficient DNA damage response (DDR) system, which is considered as a contributor to the resistance of these cells from exposures to DNA damaging agents. Targeting of enhanced DDR in CSCs is thus proposed to facilitate the eradication of CSCs by conventional therapeutics. To achieve this aim, a better understanding of the cellular responses to DNA damage in CSCs is needed. In addition to the protein kinases and enzymes that are involved in DDR, other processes that affect the DDR including chromatin remodeling should also be explored. PMID:26322164

  8. DNA Damage Response Factors from Diverse Pathways, Including DNA Crosslink Repair, Mediate Alternative End Joining

    PubMed Central

    Howard, Sean M.; Yanez, Diana A.; Stark, Jeremy M.

    2015-01-01

    Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection. PMID:25629353

  9. DNA damage response during mitosis induces whole chromosome mis-segregation

    PubMed Central

    Bakhoum, Samuel F.; Kabeche, Lilian; Murnane, John P.; Zaki, Bassem I.; Compton, Duane A.

    2014-01-01

    Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here we show that activation of the DNA damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and Plk1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or Chk2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, DDR during mitosis inappropriately stabilizes k-MTs creating a link between s-CIN and w-CIN. PMID:25107667

  10. Differential regulation of DNA damage response activation between somatic and germline cells in Caenorhabditis elegans

    PubMed Central

    Vermezovic, J; Stergiou, L; Hengartner, M O; d'Adda di Fagagna, F

    2012-01-01

    The germline of Caenorhabditis elegans is a well-established model for DNA damage response (DDR) studies. However, the molecular basis of the observed cell death resistance in the soma of these animals remains unknown. We established a set of techniques to study ionizing radiation-induced DNA damage generation and DDR activation in a whole intact worm. Our single-cell analyses reveal that, although germline and somatic cells show similar levels of inflicted DNA damage, somatic cells, differently from germline cells, do not activate the crucial apical DDR kinase ataxia-telengiectasia mutated (ATM). We also show that DDR signaling proteins are undetectable in all somatic cells and this is due to transcriptional repression. However, DNA repair genes are expressed and somatic cells retain the ability to efficiently repair DNA damage. Finally, we demonstrate that germline cells, when induced to transdifferentiate into somatic cells within the gonad, lose the ability to activate ATM. Overall, these observations provide a molecular mechanism for the known, but hitherto unexplained, resistance to DNA damage-induced cell death in C. elegans somatic cells. We propose that the observed lack of signaling and cell death but retention of DNA repair functions in the soma is a Caenorhabditis-specific evolutionary-selected strategy to cope with its lack of adult somatic stem cell pools and regenerative capacity. PMID:22705849

  11. Enhancer of rudimentary homolog regulates DNA damage response in hepatocellular carcinoma.

    PubMed

    Weng, Meng-Tzu; Tung, Tzu-Hsun; Lee, Jih-Hsiang; Wei, Shu-Chen; Lin, Hang-Li; Huang, Yu-Jung; Wong, Jau-Min; Luo, Ji; Sheu, Jin-Chuan

    2015-01-01

    We previously demonstrated that the enhancer of rudimentary homolog (ERH) gene is required for the expression of multiple cell cycle and DNA damage response (DDR) genes. The present study investigated the role of ERH and its target DNA damage repair genes in hepatocellular carcinoma cells. We observed positive correlation between ERH and ataxia telangiectasia and Rad3 related (ATR) expression in liver tissues. Expression of ERH, ATR as well as checkpoint kinase 1 (CHK1) were higher in HCCs than in normal liver tissues. Knocking-down ERH augmented ultraviolet light induced DNA damage in HepG2 cells. ATR protein level is reduced upon ERH depletion as a result of defect in the splicing of ATR mRNA. Consequently, the ATR effector kinase Chk1 failed to be phosphorylated upon ultraviolet light or hydroxyurea treatment in ERH knocked-down HepG2 cells. Finally, we observed Chk1 inhibitor AZD7762 enhanced the effect of doxorubicin on inhibiting growth of HCC cells in vitro and in vivo. This study suggested that ERH regulates the splicing of the DNA damage response proteins ATR in HCC cells, and targeting DNA damage response by Chk1 inhibitor augments chemotherapy to treat HCC cells. PMID:25880358

  12. Interplay between Np95 and Eme1 in the DNA damage response

    SciTech Connect

    Mistry, Helena; Gibson, Lianne; Yun, J.W.; Sarras, Haya; Tamblyn, Laura; McPherson, John Peter

    2008-10-24

    Mus81 (methyl methansulfonate UV sensitive clone 81) and Eme1 (essential meiotic endonuclease 1, also known as MMS4) form a heterodimeric endonuclease that is critical for genomic stability and the response to DNA crosslink damage and replication blockade. However, relatively little is known as to how this endonuclease is regulated following DNA damage. Here, we report mammalian Eme1 interacts with Np95, an E3 ubiquitin ligase that participates in chromatin modification, replication-linked epigenetic maintenance and the DNA damage response. Np95 and Eme1 co-localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. Taken together, these findings link Mus81-Eme1 with the replication-associated chromatin modifier functions of Np95 in the cellular response to DNA damage.

  13. Enhancer of rudimentary homolog regulates DNA damage response in hepatocellular carcinoma

    PubMed Central

    Weng, Meng-Tzu; Tung, Tzu-Hsun; Lee, Jih-Hsiang; Wei, Shu-Chen; Lin, Hang-Li; Huang, Yu-Jung; Wong, Jau-Min; Luo, Ji; Sheu, Jin-Chuan

    2015-01-01

    We previously demonstrated that the enhancer of rudimentary homolog (ERH) gene is required for the expression of multiple cell cycle and DNA damage response (DDR) genes. The present study investigated the role of ERH and its target DNA damage repair genes in hepatocellular carcinoma cells. We observed positive correlation between ERH and ataxia telangiectasia and Rad3 related (ATR) expression in liver tissues. Expression of ERH, ATR as well as checkpoint kinase 1 (CHK1) were higher in HCCs than in normal liver tissues. Knocking-down ERH augmented ultraviolet light induced DNA damage in HepG2 cells. ATR protein level is reduced upon ERH depletion as a result of defect in the splicing of ATR mRNA. Consequently, the ATR effector kinase Chk1 failed to be phosphorylated upon ultraviolet light or hydroxyurea treatment in ERH knocked-down HepG2 cells. Finally, we observed Chk1 inhibitor AZD7762 enhanced the effect of doxorubicin on inhibiting growth of HCC cells in vitro and in vivo. This study suggested that ERH regulates the splicing of the DNA damage response proteins ATR in HCC cells, and targeting DNA damage response by Chk1 inhibitor augments chemotherapy to treat HCC cells. PMID:25880358

  14. Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling

    PubMed Central

    Ferrando-May, Elisa; Tomas, Martin; Blumhardt, Philipp; Stöckl, Martin; Fuchs, Matthias; Leitenstorfer, Alfred

    2013-01-01

    Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments. PMID:23882280

  15. The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks.

    PubMed

    Kennedy, Daniel R; Beerman, Terry A

    2006-03-21

    Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present. PMID:16533058

  16. Lamin A/C-dependent interaction with 53BP1 promotes cellular responses to DNA damage

    PubMed Central

    Gibbs-Seymour, Ian; Markiewicz, Ewa; Bekker-Jensen, Simon; Mailand, Niels; Hutchison, Christopher J

    2015-01-01

    Lamins A/C have been implicated in DNA damage response pathways. We show that the DNA repair protein 53BP1 is a lamin A/C binding protein. In undamaged human dermal fibroblasts (HDF), 53BP1 is a nucleoskeleton protein. 53BP1 binds to lamins A/C via its Tudor domain, and this is abrogated by DNA damage. Lamins A/C regulate 53BP1 levels and consequently lamin A/C-null HDF display a 53BP1 null-like phenotype. Our data favour a model in which lamins A/C maintain a nucleoplasmic pool of 53BP1 in order to facilitate its rapid recruitment to sites of DNA damage and could explain why an absence of lamin A/C accelerates aging. PMID:25645366

  17. Protecting the heritable genome: DNA damage response mechanisms in spermatogonial stem cells.

    PubMed

    Rübe, Claudia E; Zhang, Sheng; Miebach, Nadine; Fricke, Andreas; Rübe, Christian

    2011-02-01

    Spermatogonial stem cells (SSCs) must maintain the integrity of their genome to prevent reproduction failure and limit the hereditary risk associated with transmission to the progeny. SSCs must therefore have robust response mechanisms to counteract the potentially deleterious effects of DNA damage, with DNA double-strand breaks (DSBs) representing the greatest threat to genomic integrity. Through in vivo analysis of the DNA damage response of SSCs within their physiological tissue context, we aimed to gain insights into the mechanisms by which SSCs preserve genome integrity. After whole-body irradiation of repair-proficient and repair-deficient (DNA-PK- and ATM-deficient) mice, the formation and rejoining of DSBs was analyzed in SSCs of testis compared with somatic cells of other tissues by enumerating γH2AX-, MDC1-, and 53BP1-foci. Caspase-3 and PARP-1 were used as markers for apoptotic cell death. Our results show that DNA damage response mechanisms in SSCs characterized by unique chromatin compositions are markedly different from those of somatic cells. In SSCs lacking compact heterochromatin, histone-associated signaling components of the DNA repair machinery are completely absent and radiation-induced DSBs are rejoined predominantly by DNA-PK-independent pathways, suggesting the existence of alternative repair mechanisms. As a complimentary mechanism characterized by low thresholds for ATM-dependent checkpoint activation, the differentiating progeny, but not the SSCs themselves, promote apoptosis in response to low levels of DNA damage. By evaluating SSCs within their stem cell niche, we show that DNA repair, cell-cycle checkpoints, and apoptosis function together to maintain the integrity of the heritable genome. PMID:21123119

  18. FBXO31: a new player in the ever-expanding DNA damage response orchestra.

    PubMed

    Shiloh, Yosef

    2009-01-01

    The DNA damage response (DDR)-a central axis in the maintenance of genomic stability-has emerged as a complex signaling network that affects many aspects of cellular metabolism. A major arm of the DDR activates special checkpoints that temporarily arrest cell cycle progression while damage is being assessed and processed. Many DDR arms are driven by several parallel pathways acting in concert. Such is the case with the damage-induced G(1)/S checkpoint. A new pathway driving this checkpoint draws attention to the complexity of the DDR, which allows tight but fine-tuned control of the cellular response to threats to genomic integrity. PMID:19903939

  19. Molecular Pathways: Targeting the Dependence of Mutant RAS Cancers on the DNA Damage Response

    PubMed Central

    Grabocka, Elda; Commisso, Cosimo; Bar-Sagi, Dafna

    2014-01-01

    Of the genes mutated in cancer, RAS remains the most elusive to target. Recent technological advances and discoveries have greatly expanded our knowledge of the biology of oncogenic Ras and its role in cancer. As such, it has become apparent that a property that intimately accompanies RAS-driven tumorigenesis is the dependence of RAS mutant cells on a number of non-oncogenic signaling pathways. These dependencies arise as a means of adaptation to Ras-driven intracellular stresses and represent unique vulnerabilities of mutant RAS cancers. A number of studies have highlighted the dependence of mutant RAS cancers on the DNA damage response and identified the molecular pathways that mediate this process including signaling from wild-type Ras isoforms, ATR/Chk1, and DNA damage repair pathways. Here we review these findings, and discuss the combinatorial use of DNA damaging chemotherapy with blockade of wild-type H- and N-Ras signaling by farnesyltransferase inhibitors, Chk1 inhibitors, or small molecule targeting DNA damage repair as potential strategies through which the dependence of RAS cancers on the DNA damage response can be harnessed for therapeutic intervention. PMID:25424849

  20. Clofarabine Acts as Radiosensitizer In Vitro and In Vivo by Interfering With DNA Damage Response

    SciTech Connect

    Cariveau, Mickael J.; Stackhouse, Murray; Cui Xiaoli; Tiwari, Kamal; Waud, William; Secrist, John A.; Xu Bo

    2008-01-01

    Purpose: Combination treatment with radiotherapy and chemotherapy has emerged as the dominant form of cancer adjuvant regimens in recent years. Clofarabine, a newly approved drug for pediatric leukemia, is a second-generation purine nucleoside analogue that can block DNA synthesis and inhibit DNA repair. Therefore, we hypothesized that clofarabine could work synergistically with radiotherapy to increase the tumor cell response. Methods and Materials: The effects of clofarabine on radiosensitivity have been established in several tumor cell lines in vitro and in vivo using colony-forming assays and tumor xenografts. The effect of clofarabine on the DNA damage response was also studied in vitro by measuring {gamma}-H2AX focus formation. Results: Clonogenic survival was significantly reduced in irradiated cells treated with clofarabine, demonstrating the strong radiosensitizing effect of clofarabine. Furthermore, clofarabine displayed a radiosensitizing effect that was greater than gemcitabine or 5-fluorouracil. We also found that low doses of clofarabine can prolong the presence of radiation-induced {gamma}-H2AX nuclear focus formation, and high doses of clofarabine can induce DNA double-strand breaks, suggesting that clofarabine can interfere with DNA damage response pathways. In addition, clofarabine-induced radiosensitization was also established in vivo using a colorectal cancer model, DLD-1, in athymic nude mice. When combined with fractionated radiotherapy, a moderate dose of clofarabine led to a significant increase in tumor growth inhibition. Conclusion: Clofarabine acts as a powerful radiosensitizer both in vitro and in vivo by interfering with the DNA damage response.

  1. Hyperactivation of ATM upon DNA-PKcs inhibition modulates p53 dynamics and cell fate in response to DNA damage.

    PubMed

    Finzel, Ana; Grybowski, Andrea; Strasen, Jette; Cristiano, Elena; Loewer, Alexander

    2016-08-01

    A functional DNA damage response is essential for maintaining genome integrity in the presence of DNA double-strand breaks. It is mainly coordinated by the kinases ATM, ATR, and DNA-PKcs, which control the repair of broken DNA strands and relay the damage signal to the tumor suppressor p53 to induce cell cycle arrest, apoptosis, or senescence. Although many functions of the individual kinases have been identified, it remains unclear how they act in concert to ensure faithful processing of the damage signal. Using specific inhibitors and quantitative analysis at the single-cell level, we systematically characterize the contribution of each kinase for regulating p53 activity. Our results reveal a new regulatory interplay in which loss of DNA-PKcs function leads to hyperactivation of ATM and amplification of the p53 response, sensitizing cells for damage-induced senescence. This interplay determines the outcome of treatment regimens combining irradiation with DNA-PKcs inhibitors in a p53-dependent manner. PMID:27280387

  2. Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology.

    PubMed

    Grasso, Francesca; Frisan, Teresa

    2015-01-01

    Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins. PMID:26270677

  3. Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology

    PubMed Central

    Grasso, Francesca; Frisan, Teresa

    2015-01-01

    Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins. PMID:26270677

  4. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    NASA Astrophysics Data System (ADS)

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  5. Nek4 Regulates Entry into Replicative Senescence and the Response to DNA Damage in Human Fibroblasts

    PubMed Central

    Nguyen, Christine L.; Possemato, Richard; Bauerlein, Erica L.; Xie, Anyong; Scully, Ralph

    2012-01-01

    When explanted into culture, normal human cells exhibit a finite number of cell divisions before entering a proliferative arrest termed replicative senescence. To identify genes essential for entry into replicative senescence, we performed an RNA interference (RNAi)-based loss-of-function screen and found that suppression of the Never in Mitosis Gene A (NIMA)-related protein kinase gene NEK4 disrupted timely entry into senescence. NEK4 suppression extended the number of population doublings required to reach replicative senescence in several human fibroblast strains and resulted in decreased transcription of the cyclin-dependent kinase inhibitor p21. NEK4-suppressed cells displayed impaired cell cycle arrest in response to double-stranded DNA damage, and mass spectrometric analysis of Nek4 immune complexes identified a complex containing DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)], Ku70, and Ku80. NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to DNA upon induction of double-stranded DNA damage, resulting in reduced p53 activation and H2AX phosphorylation. Together, these observations implicate Nek4 as a novel regulator of replicative senescence and the response to double-stranded DNA damage. PMID:22851694

  6. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  7. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B

    PubMed Central

    Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  8. DNA Damage Response and Spindle Assembly Checkpoint Function throughout the Cell Cycle to Ensure Genomic Integrity

    PubMed Central

    Lawrence, Katherine S.; Chau, Thinh; Engebrecht, JoAnne

    2015-01-01

    Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved. PMID:25898113

  9. Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

    PubMed

    Klapacz, Joanna; Pottenger, Lynn H; Engelward, Bevin P; Heinen, Christopher D; Johnson, George E; Clewell, Rebecca A; Carmichael, Paul L; Adeleye, Yeyejide; Andersen, Melvin E

    2016-01-01

    From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. PMID:27036068

  10. Role of c-Abl in the DNA damage stress response.

    PubMed

    Shaul, Yosef; Ben-Yehoyada, Merav

    2005-01-01

    c-Abl has been implicated in many cellular processes including differentiation, division, adhesion, death, and stress response. c-Abl is a latent tyrosine kinase that becomes activated in response to numerous extra- and intra-cellular stimuli. Here we briefly review the current knowledge about c-Abl involvement in the DNA-damage stress response and its implication on cell physiology. PMID:15686624

  11. Nitric Oxide Suppresses β-Cell Apoptosis by Inhibiting the DNA Damage Response.

    PubMed

    Oleson, Bryndon J; Broniowska, Katarzyna A; Naatz, Aaron; Hogg, Neil; Tarakanova, Vera L; Corbett, John A

    2016-08-01

    Nitric oxide, produced in pancreatic β cells in response to proinflammatory cytokines, plays a dual role in the regulation of β-cell fate. While nitric oxide induces cellular damage and impairs β-cell function, it also promotes β-cell survival through activation of protective pathways that promote β-cell recovery. In this study, we identify a novel mechanism in which nitric oxide prevents β-cell apoptosis by attenuating the DNA damage response (DDR). Nitric oxide suppresses activation of the DDR (as measured by γH2AX formation and the phosphorylation of KAP1 and p53) in response to multiple genotoxic agents, including camptothecin, H2O2, and nitric oxide itself, despite the presence of DNA damage. While camptothecin and H2O2 both induce DDR activation, nitric oxide suppresses only camptothecin-induced apoptosis and not H2O2-induced necrosis. The ability of nitric oxide to suppress the DDR appears to be selective for pancreatic β cells, as nitric oxide fails to inhibit DDR signaling in macrophages, hepatocytes, and fibroblasts, three additional cell types examined. While originally described as the damaging agent responsible for cytokine-induced β-cell death, these studies identify a novel role for nitric oxide as a protective molecule that promotes β-cell survival by suppressing DDR signaling and attenuating DNA damage-induced apoptosis. PMID:27185882

  12. Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes

    PubMed Central

    Mollet, Inês G.; Giess, Adam; Paschalaki, Koralia; Periyasamy, Manikandan; Lidington, Elaine C.; Mason, Justin C.; Jones, Michael D.; Game, Laurence; Ali, Simak; Shovlin, Claire L.

    2016-01-01

    Background Spontaneous reports from patients able to report vascular sequelae in real time, and recognition that serum non transferrin bound iron may reach or exceed 10μmol/L in the blood stream after iron tablets or infusions, led us to hypothesize that conventional iron treatments may provoke acute vascular injury. This prompted us to examine whether a phenotype could be observed in normal human endothelial cells treated with low dose iron. Methodology Confluent primary human endothelial cells (EC) were treated with filter-sterilized iron (II) citrate or fresh media for RNA sequencing and validation studies. RNA transcript profiles were evaluated using directional RNA sequencing with no pre-specification of target sequences. Alignments were counted for exons and junctions of the gene strand only, blinded to treatment types. Principal Findings Rapid changes in RNA transcript profiles were observed in endothelial cells treated with 10μmol/L iron (II) citrate, compared to media-treated cells. Clustering for Gene Ontology (GO) performed on all differentially expressed genes revealed significant differences in biological process terms between iron and media-treated EC, whereas 10 sets of an equivalent number of randomly selected genes from the respective EC gene datasets showed no significant differences in any GO terms. After 1 hour, differentially expressed genes clustered to vesicle mediated transport, protein catabolism, and cell cycle (Benjamini p = 0.0016, 0.0024 and 0.0032 respectively), and by 6 hours, to cellular response to DNA damage stimulus most significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10μM iron treatment elicited DNA damage within 1 hour. This was accompanied by a brisk DNA damage response pulse, as ascertained by the development of DNA damage response (DDR) foci, and p53 stabilization. Significance These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium

  13. Inhibiting the DNA damage response as a therapeutic manoeuvre in cancer

    PubMed Central

    Curtin, N J

    2013-01-01

    The DNA damage response (DDR), consisting of an orchestrated network of proteins effecting repair and signalling to cell cycle arrest, to allow time to repair, is essential for cell viability and to prevent DNA damage being passed on to daughter cells. The DDR is dysregulated in cancer with some pathways up-regulated and others down-regulated or lost. Up-regulated pathways can confer resistance to anti-cancer DNA damaging agents. Therefore, inhibitors of key components of these pathways have the potential to prevent this therapeutic resistance. Conversely, defects in a particular DDR pathway may lead to dependence on a complementary pathway. Inhibition of this complementary pathway may result in tumour-specific cell killing. Thus, inhibitors of the DDR have the potential to increase the efficacy of DNA damaging chemotherapy and radiotherapy and have single-agent activity against tumours with a specific DDR defect. This review describes the compounds that have been designed to inhibit specific DDR targets and summarizes the pre-clinical and clinical evaluation of these inhibitors of DNA damage signalling and repair. Linked Articles This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8 PMID:23682925

  14. SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells

    PubMed Central

    Murakami-Tonami, Yuko; Ikeda, Haruna; Yamagishi, Ryota; Inayoshi, Mao; Inagaki, Shiho; Kishida, Satoshi; Komata, Yosuke; Jan Koster, J K; Takeuchi, Ichiro; Kondo, Yutaka; Maeda, Tohru; Sekido, Yoshitaka; Murakami, Hiroshi; Kadomatsu, Kenji

    2016-01-01

    Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of SGO1 impaired proliferation and induced DNA damage followed by a senescence-like phenotype only in MYCN-overexpressing neuroblastoma cells. In these cells, SGO1 knockdown induced DNA damage, even during interphase, and this effect was independent of cohesin. Furthermore, MYCN-promoted SGO1 transcription and SGO1 expression tended to be higher in MYCN- or MYC-overexpressing cancers. Together, these findings indicate that SGO1 plays a role in the DNA damage response in interphase. Therefore, we propose that SGO1 represents a potential molecular target for treatment of MYCN-amplified neuroblastoma. PMID:27539729

  15. SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells.

    PubMed

    Murakami-Tonami, Yuko; Ikeda, Haruna; Yamagishi, Ryota; Inayoshi, Mao; Inagaki, Shiho; Kishida, Satoshi; Komata, Yosuke; Jan Koster; Takeuchi, Ichiro; Kondo, Yutaka; Maeda, Tohru; Sekido, Yoshitaka; Murakami, Hiroshi; Kadomatsu, Kenji

    2016-01-01

    Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of SGO1 impaired proliferation and induced DNA damage followed by a senescence-like phenotype only in MYCN-overexpressing neuroblastoma cells. In these cells, SGO1 knockdown induced DNA damage, even during interphase, and this effect was independent of cohesin. Furthermore, MYCN-promoted SGO1 transcription and SGO1 expression tended to be higher in MYCN- or MYC-overexpressing cancers. Together, these findings indicate that SGO1 plays a role in the DNA damage response in interphase. Therefore, we propose that SGO1 represents a potential molecular target for treatment of MYCN-amplified neuroblastoma. PMID:27539729

  16. S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

    SciTech Connect

    Lee, Haemi; Kwak, Hee-Jin; Cho, Il-taeg; Park, Seok Hee; Lee, Chang-Hun

    2009-01-02

    53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and {gamma}H2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.

  17. Filia is an ESC-specific regulator of DNA damage response and safeguards genomic stability

    PubMed Central

    Zhao, Bo; Zhang, Wei-dao; Duan, Ying-liang; Lu, Yong-qing; Cun, Yi-xian; Li, Chao-hui; Guo, Kun; Nie, Wen-hui; Li, Lei; Zhang, Rugang; Zheng, Ping

    2015-01-01

    Summary Pluripotent stem cells (PSCs) hold great promise in cell-based therapy, but the genomic instability seen in culture hampers full application. Greater understanding of the factors that regulate genomic stability in PSCs could help address this issue. Here we describe the identification of Filia as a specific regulator of genomic stability in mouse embryonic stem cells (ESCs). Filia expression is induced by genotoxic stress. Filia promotes centrosome integrity and regulates DNA damage response (DDR) through multiple pathways, including DDR signaling, cell cycle checkpoints and damage repair, ESC differentiation and apoptosis. Filia depletion causes ESC genomic instability, induces resistance to apoptosis and promotes malignant transformation. As part of its role in the DDR, Filia interacts with PARP1 and stimulates its enzymatic activity. Filia also constitutively resides on centrosomes and translocates to DNA damage sites and mitochondria, consistent with its multifaceted roles in regulating centrosome integrity, damage repair and apoptosis. PMID:25936915

  18. Variable Activation of the DNA Damage Response Pathways in Patients Undergoing SPECT Myocardial Perfusion Imaging

    PubMed Central

    Hu, Shijun; Liang, Grace; Ong, Sang-Ging; Han, Leng; Sanchez-Freire, Veronica; Lee, Andrew S.; Vasanawala, Minal; Segall, George; Wu, Joseph C.

    2015-01-01

    Background Although single photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low dose radiation exposure for cardiac patients. To determine the biological effects of low dose radiation from SPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single cell gene expression profiling. Methods and Results Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, p53, and ATM) following SPECT. The majority of patients also had either down-regulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation also had significant up-regulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant down-regulation or no difference, suggesting that some patients may potentially be more sensitive to low dose radiation exposure. Conclusions Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression post-imaging, continued care should be taken to reduce radiation exposure to both patients and operators. PMID:25609688

  19. At the Bench: Helicobacter pylori, dysregulated host responses, DNA damage, and gastric cancer.

    PubMed

    Hardbower, Dana M; Peek, Richard M; Wilson, Keith T

    2014-08-01

    Helicobacter pylori infection is the strongest known risk factor for the development of gastric cancer. Given that ∼50% of the global population is infected with this pathogen, there is great impetus to elucidate underlying causes that mediate progression from infection to cancer. Recent evidence suggests that H. pylori-induced chronic inflammation and oxidative stress create an environment conducive to DNA damage and tissue injury. DNA damage leads to genetic instability and eventually, neoplastic transformation. Pathogen-encoded virulence factors induce a robust but futile immune response and alter host pathways that lower the threshold for carcinogenesis, including DNA damage repair, polyamine synthesis and catabolism, antioxidant responses, and cytokine production. Collectively, such dysregulation creates a protumorigenic microenvironment within the stomach. This review seeks to address each of these aspects of H. pylori infection and to call attention to areas of particular interest within this field of research. This review also seeks to prioritize areas of translational research related to H. pylori-induced gastric cancer based on insights garnered from basic research in this field. See related review by Dalal and Moss, At the Bedside: H. pylori, dysregulated host responses, DNA damage, and gastric cancer. PMID:24868089

  20. At the Bench: Helicobacter pylori, dysregulated host responses, DNA damage, and gastric cancer

    PubMed Central

    Hardbower, Dana M.; Peek, Richard M.; Wilson, Keith T.

    2014-01-01

    Helicobacter pylori infection is the strongest known risk factor for the development of gastric cancer. Given that ∼50% of the global population is infected with this pathogen, there is great impetus to elucidate underlying causes that mediate progression from infection to cancer. Recent evidence suggests that H. pylori-induced chronic inflammation and oxidative stress create an environment conducive to DNA damage and tissue injury. DNA damage leads to genetic instability and eventually, neoplastic transformation. Pathogen-encoded virulence factors induce a robust but futile immune response and alter host pathways that lower the threshold for carcinogenesis, including DNA damage repair, polyamine synthesis and catabolism, antioxidant responses, and cytokine production. Collectively, such dysregulation creates a protumorigenic microenvironment within the stomach. This review seeks to address each of these aspects of H. pylori infection and to call attention to areas of particular interest within this field of research. This review also seeks to prioritize areas of translational research related to H. pylori-induced gastric cancer based on insights garnered from basic research in this field. See related review by Dalal and Moss, At the Bedside: H. pylori, dysregulated host responses, DNA damage, and gastric cancer. PMID:24868089

  1. Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response.

    PubMed

    Yogev, Ohad; Yogev, Orli; Singer, Esti; Shaulian, Eitan; Goldberg, Michal; Fox, Thomas D; Pines, Ophry

    2010-03-01

    In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation

  2. Targeting the epigenetics of the DNA damage response in breast cancer

    PubMed Central

    Montenegro, M F; González-Guerrero, R; Sánchez-del-Campo, L; Piñero-Madrona, A; Cabezas-Herrera, J; Rodríguez-López, J N

    2016-01-01

    Cancer is as much an epigenetic disease as it is a genetic disease, and epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Because the epigenetic factors involved in the DNA damage response are regulated by multiple elements, therapies to target specific components of the epigenetic machinery can be inefficient. In contrast, therapies aimed at inhibiting the methionine cycle can indirectly inhibit both DNA and protein methylation, and the wide variety of genes and pathways that are affected by these methylations make this global strategy very attractive. In the present study, we propose an adjuvant therapy that targets the epigenetics of the DNA damage response in breast cancer cells and that results in efficient apoptosis and a reduction in distant metastases in vivo. We observed that a combined therapy designed to uncouple adenosine metabolism using dipyridamole in the presence of a new synthetic antifolate, 3-O-(3,4,5-trimethoxybenzoyl)-(−)-catechin, simultaneously and efficiently blocked both the folic cycle and the methionine cycle in breast cancer cells and sensitized these cells to radiotherapy. The treatment impeded the recruitment of 53BP1 and BRCA1 to the chromatin regions flanking DNA double-strand breaks and thereby avoided the DNA damage responses in breast cancer cells that were exposed to ionizing radiation. In addition, this hypomethylating therapy was also efficient in reducing the self-renewal capability of breast cancer-initiating cells and induced reversion of mesenchymal phenotypes in breast cancer cells. PMID:27054335

  3. Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

    PubMed

    Mavuluri, Jayadev; Beesetti, Swarnalatha; Surabhi, Rohan; Kremerskothen, Joachim; Venkatraman, Ganesh; Rayala, Suresh K

    2016-05-01

    Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential

  4. Activation of DNA damage repair pathways in response to nitrogen mustard-induced DNA damage and toxicity in skin keratinocytes.

    PubMed

    Inturi, Swetha; Tewari-Singh, Neera; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

    2014-01-01

    Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury. PMID:24732344

  5. Activation of DNA damage repair pathways in response to nitrogen mustard-induced DNA damage and toxicity in skin keratinocytes

    PubMed Central

    Inturi, Swetha; Tewari-Singh, Neera; Agarwal, Chapla; White, Carl W.; Agarwal, Rajesh

    2014-01-01

    Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury. PMID:24732344

  6. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  7. SOG1: a master regulator of the DNA damage response in plants.

    PubMed

    Yoshiyama, Kaoru Okamoto

    2016-01-01

    The DNA damage response (DDR) is a critical mechanism to maintain the genome stability of an organism upon exposure to endogenous and exogenous DNA-damaging factors. The DDR system is particularly important for plants as these organisms, owing to their intrinsic immobility, are inevitably exposed to environmental stress factors, some of which induce DNA damage. Arabidopsis thaliana has orthologs of several DDR factors that are present in animals; however, some of the important animal regulators, such as the tumor suppressor p53 and the DDR kinases CHK1 and CHK2, have not been found in plants. These observations imply a unique DDR system in plants. The present review focuses on recent advances in our understanding of the DDR in A. thaliana and, in particular, on the function and role of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a plant-specific transcription factor that regulates the DDR. The most obvious response to DNA damage in A. thaliana is a rapid and robust change in the transcriptional regulation of numerous genes, in which SOG1 is an essential regulatory factor. Mutation of SOG1 causes various defects in the activation of cell cycle arrest, programmed cell death, and endoreduplication in response to DNA damage. These observations indicate that SOG1 is a master regulator of the DDR. Phylogenetic analyses of SOG1 reveal that orthologs of this crucial transcription factor are present not only in angiosperms but also in gymnosperms, suggesting that the SOG1 system is conserved across spermatophytes. Finally, future prospects for SOG1 research are also discussed. PMID:26617076

  8. NMR metabolomic profiling reveals new roles of SUMOylation in DNA damage response.

    PubMed

    Cano, Kristin E; Li, Yi-Jia; Chen, Yuan

    2010-10-01

    Post-translational modifications by the Small Ubiquitin-like Modifier (SUMO) family of proteins have been established as critical events in the cellular response to a wide range of DNA damaging reagents and radiation; however, the detailed mechanism of SUMOylation in DNA damage response is not well understood. In this study, we used a nuclear magnetic resonance (NMR) spectroscopy-based metabolomics approach to examine the effect of an inhibitor of SUMO-mediated protein-protein interactions on MCF7 breast cancer cell response to radiation. Metabolomics is sensitive to changes in cellular functions and thus provides complementary information to other biological studies. The peptide inhibitor (SUMO interaction motif mimic, SIM) and a control peptide were stably expressed in MCF-7 cell line. Metabolite profiles of the cell lines before and after radiation were analyzed using solution NMR methods. Various statistical methods were used to isolate significant changes. Differences in the amounts of glutamine, aspartate, malate, alanine, glutamate and NADH between the SIM-expressing and control cells suggest a role for SUMOylation in regulating mitochondrial function. This is also further verified following the metabolism of (13)C-labeled glutamine. The inability of the cells expressing the SIM peptide to increase production of the antioxidants carnosine and glutathione after radiation damage suggests an important role of SUMOylation in regulating the levels of antioxidants that protect cells from free radicals and reactive oxygen species generated by radiation. This study reveals previously unknown roles of SUMOylation in DNA damage response. PMID:20695451

  9. ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response.

    PubMed

    Guo, Yingying; Feng, Wanjuan; Sy, Shirley M H; Huen, Michael S Y

    2015-11-13

    The Fanconi anemia protein PALB2, also known as FANCN, protects genome integrity by regulating DNA repair and cell cycle checkpoints. Exactly how PALB2 functions may be temporally coupled with detection and signaling of DNA damage is not known. Intriguingly, we found that PALB2 is transformed into a hyperphosphorylated state in response to ionizing radiation (IR). IR treatment specifically triggered PALB2 phosphorylation at Ser-157 and Ser-376 in manners that required the master DNA damage response kinase Ataxia telangiectasia mutated, revealing potential mechanistic links between PALB2 and the Ataxia telangiectasia mutated-dependent DNA damage responses. Consistently, dysregulated PALB2 phosphorylation resulted in sustained activation of DDRs. Full-blown PALB2 phosphorylation also required the breast and ovarian susceptible gene product BRCA1, highlighting important roles of the BRCA1-PALB2 interaction in orchestrating cellular responses to genotoxic stress. In summary, our phosphorylation analysis of tumor suppressor protein PALB2 uncovers new layers of regulatory mechanisms in the maintenance of genome stability and tumor suppression. PMID:26420486

  10. Predicted Role of NAD Utilization in the Control of Circadian Rhythms during DNA Damage Response

    PubMed Central

    Luna, Augustin; McFadden, Geoffrey B.; Aladjem, Mirit I.; Kohn, Kurt W.

    2015-01-01

    The circadian clock is a set of regulatory steps that oscillate with a period of approximately 24 hours influencing many biological processes. These oscillations are robust to external stresses, and in the case of genotoxic stress (i.e. DNA damage), the circadian clock responds through phase shifting with primarily phase advancements. The effect of DNA damage on the circadian clock and the mechanism through which this effect operates remains to be thoroughly investigated. Here we build an in silico model to examine damage-induced circadian phase shifts by investigating a possible mechanism linking circadian rhythms to metabolism. The proposed model involves two DNA damage response proteins, SIRT1 and PARP1, that are each consumers of nicotinamide adenine dinucleotide (NAD), a metabolite involved in oxidation-reduction reactions and in ATP synthesis. This model builds on two key findings: 1) that SIRT1 (a protein deacetylase) is involved in both the positive (i.e. transcriptional activation) and negative (i.e. transcriptional repression) arms of the circadian regulation and 2) that PARP1 is a major consumer of NAD during the DNA damage response. In our simulations, we observe that increased PARP1 activity may be able to trigger SIRT1-induced circadian phase advancements by decreasing SIRT1 activity through competition for NAD supplies. We show how this competitive inhibition may operate through protein acetylation in conjunction with phosphorylation, consistent with reported observations. These findings suggest a possible mechanism through which multiple perturbations, each dominant during different points of the circadian cycle, may result in the phase advancement of the circadian clock seen during DNA damage. PMID:26020938

  11. Predicted Role of NAD Utilization in the Control of Circadian Rhythms during DNA Damage Response.

    PubMed

    Luna, Augustin; McFadden, Geoffrey B; Aladjem, Mirit I; Kohn, Kurt W

    2015-05-01

    The circadian clock is a set of regulatory steps that oscillate with a period of approximately 24 hours influencing many biological processes. These oscillations are robust to external stresses, and in the case of genotoxic stress (i.e. DNA damage), the circadian clock responds through phase shifting with primarily phase advancements. The effect of DNA damage on the circadian clock and the mechanism through which this effect operates remains to be thoroughly investigated. Here we build an in silico model to examine damage-induced circadian phase shifts by investigating a possible mechanism linking circadian rhythms to metabolism. The proposed model involves two DNA damage response proteins, SIRT1 and PARP1, that are each consumers of nicotinamide adenine dinucleotide (NAD), a metabolite involved in oxidation-reduction reactions and in ATP synthesis. This model builds on two key findings: 1) that SIRT1 (a protein deacetylase) is involved in both the positive (i.e. transcriptional activation) and negative (i.e. transcriptional repression) arms of the circadian regulation and 2) that PARP1 is a major consumer of NAD during the DNA damage response. In our simulations, we observe that increased PARP1 activity may be able to trigger SIRT1-induced circadian phase advancements by decreasing SIRT1 activity through competition for NAD supplies. We show how this competitive inhibition may operate through protein acetylation in conjunction with phosphorylation, consistent with reported observations. These findings suggest a possible mechanism through which multiple perturbations, each dominant during different points of the circadian cycle, may result in the phase advancement of the circadian clock seen during DNA damage. PMID:26020938

  12. Mechanisms of DNA damage response to targeted irradiation in organotypic 3D skin cultures.

    PubMed

    Acheva, Anna; Ghita, Mihaela; Patel, Gaurang; Prise, Kevin M; Schettino, Giuseppe

    2014-01-01

    DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2. PMID:24505255

  13. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    PubMed

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process. PMID:27148355

  14. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks

    PubMed Central

    Dantuma, Nico P.; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process. PMID:27148355

  15. Complex interactions between the DNA-damage response and mammalian telomeres

    PubMed Central

    Arnoult, Nausica; Karlseder, Jan

    2016-01-01

    Natural chromosome ends resemble double-stranded DNA breaks, but they do not activate a damage response in healthy cells. Telomeres therefore have evolved to solve the ‘end-protection problem’ by inhibiting multiple DNA damage–response pathways. During the past decade, the view of telomeres has progressed from simple caps that hide chromosome ends to complex machineries that have an active role in organizing the genome. Here we focus on mammalian telomeres and summarize and interpret recent discoveries in detail, focusing on how repair pathways are inhibited, how resection and replication are controlled and how these mechanisms govern cell fate during senescence, crisis and transformation. PMID:26581520

  16. The Adaptive Significance of Natural Genetic Variation in the DNA Damage Response of Drosophila melanogaster

    PubMed Central

    Svetec, Nicolas; Cridland, Julie M.; Zhao, Li; Begun, David J.

    2016-01-01

    Despite decades of work, our understanding of the distribution of fitness effects of segregating genetic variants in natural populations remains largely incomplete. One form of selection that can maintain genetic variation is spatially varying selection, such as that leading to latitudinal clines. While the introduction of population genomic approaches to understanding spatially varying selection has generated much excitement, little successful effort has been devoted to moving beyond genome scans for selection to experimental analysis of the relevant biology and the development of experimentally motivated hypotheses regarding the agents of selection; it remains an interesting question as to whether the vast majority of population genomic work will lead to satisfying biological insights. Here, motivated by population genomic results, we investigate how spatially varying selection in the genetic model system, Drosophila melanogaster, has led to genetic differences between populations in several components of the DNA damage response. UVB incidence, which is negatively correlated with latitude, is an important agent of DNA damage. We show that sensitivity of early embryos to UVB exposure is strongly correlated with latitude such that low latitude populations show much lower sensitivity to UVB. We then show that lines with lower embryo UVB sensitivity also exhibit increased capacity for repair of damaged sperm DNA by the oocyte. A comparison of the early embryo transcriptome in high and low latitude embryos provides evidence that one mechanism of adaptive DNA repair differences between populations is the greater abundance of DNA repair transcripts in the eggs of low latitude females. Finally, we use population genomic comparisons of high and low latitude samples to reveal evidence that multiple components of the DNA damage response and both coding and non-coding variation likely contribute to adaptive differences in DNA repair between populations. PMID:26950216

  17. The Adaptive Significance of Natural Genetic Variation in the DNA Damage Response of Drosophila melanogaster.

    PubMed

    Svetec, Nicolas; Cridland, Julie M; Zhao, Li; Begun, David J

    2016-03-01

    Despite decades of work, our understanding of the distribution of fitness effects of segregating genetic variants in natural populations remains largely incomplete. One form of selection that can maintain genetic variation is spatially varying selection, such as that leading to latitudinal clines. While the introduction of population genomic approaches to understanding spatially varying selection has generated much excitement, little successful effort has been devoted to moving beyond genome scans for selection to experimental analysis of the relevant biology and the development of experimentally motivated hypotheses regarding the agents of selection; it remains an interesting question as to whether the vast majority of population genomic work will lead to satisfying biological insights. Here, motivated by population genomic results, we investigate how spatially varying selection in the genetic model system, Drosophila melanogaster, has led to genetic differences between populations in several components of the DNA damage response. UVB incidence, which is negatively correlated with latitude, is an important agent of DNA damage. We show that sensitivity of early embryos to UVB exposure is strongly correlated with latitude such that low latitude populations show much lower sensitivity to UVB. We then show that lines with lower embryo UVB sensitivity also exhibit increased capacity for repair of damaged sperm DNA by the oocyte. A comparison of the early embryo transcriptome in high and low latitude embryos provides evidence that one mechanism of adaptive DNA repair differences between populations is the greater abundance of DNA repair transcripts in the eggs of low latitude females. Finally, we use population genomic comparisons of high and low latitude samples to reveal evidence that multiple components of the DNA damage response and both coding and non-coding variation likely contribute to adaptive differences in DNA repair between populations. PMID:26950216

  18. RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

    PubMed Central

    Maréchal, Alexandre; Zou, Lee

    2015-01-01

    The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

  19. Autophagy Regulates Chromatin Ubiquitination in DNA Damage Response through Elimination of SQSTM1/p62.

    PubMed

    Wang, Yanan; Zhang, Nan; Zhang, Luyao; Li, Ran; Fu, Wan; Ma, Ke; Li, Xue; Wang, Lina; Wang, Jiadong; Zhang, Hongquan; Gu, Wei; Zhu, Wei-Guo; Zhao, Ying

    2016-07-01

    Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, and loss of autophagy has been linked to increased genome instability. Here, we report that loss of autophagy is coupled to reduced histone H2A ubiquitination after DNA damage. p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, an E3 ligase essential for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSB repair. Moreover, nuclear-localized p62 increased the sensitivity of tumor cells to radiation both in vitro and in vivo, and this required its interaction with RNF168. Our findings indicate that autophagy-deficiency-induced p62 accumulation results in inhibition of histone ubiquitination and highlight the complex relationship between autophagy and the DNA damage response. PMID:27345151

  20. Nucleolar exit of RNF8 and BRCA1 in response to DNA damage

    SciTech Connect

    Guerra-Rebollo, Marta; Mateo, Francesca; Franke, Kristin; Huen, Michael S.Y.; Lopitz-Otsoa, Fernando; Rodriguez, Manuel S.; Plans, Vanessa; Thomson, Timothy M.

    2012-11-01

    The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to {gamma}-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did {gamma}-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage. -- Highlights: Black-Right-Pointing-Pointer RNF8 and BRCA1 are associated with the nucleolus of undamaged cells. Black-Right-Pointing-Pointer Upon {gamma}-radiation, RNF8 and BRCA1 are translocated from the nucleolus to damage foci. Black-Right-Pointing-Pointer The ribosomal protein RPSA anchors RNF8 to the nucleolus. Black-Right-Pointing-Pointer RNF8 may play previously unsuspected roles in protein synthesis.

  1. DAF-16/FoxO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage

    PubMed Central

    Babu, Vipin; Ermolaeva, Maria A.; Müller, Roman-Ulrich; Frommolt, Peter; Williams, Ashley B.; Greiss, Sebastian; Schneider, Jennifer I.; Benzing, Thomas; Schermer, Bernhard; Schumacher, Björn

    2014-01-01

    Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility, and premature aging. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with aging. Here we show that the FoxO transcription factor DAF-16 is activated in response to DNA damage during development while the DNA damage responsiveness of DAF-16 declines with aging. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16 mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists. PMID:25419847

  2. Anthracyclines Induce DNA Damage Response-Mediated Protection against Severe Sepsis

    PubMed Central

    Figueiredo, Nuno; Chora, Angelo; Raquel, Helena; Pejanovic, Nadja; Pereira, Pedro; Hartleben, Björn; Neves-Costa, Ana; Moita, Catarina; Pedroso, Dora; Pinto, Andreia; Marques, Sofia; Faridi, Hafeez; Costa, Paulo; Gozzelino, Raffaella; Zhao, Jimmy L.; Soares, Miguel P.; Gama-Carvalho, Margarida; Martinez, Jennifer; Zhang, Qingshuo; Döring, Gerd; Grompe, Markus; Simas, J. Pedro; Huber, Tobias B.; Baltimore, David; Gupta, Vineet; Green, Douglas R.; Ferreira, João A.; Moita, Luis F.

    2014-01-01

    Summary Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fancony Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis. PMID:24184056

  3. Anthracyclines induce DNA damage response-mediated protection against severe sepsis.

    PubMed

    Figueiredo, Nuno; Chora, Angelo; Raquel, Helena; Pejanovic, Nadja; Pereira, Pedro; Hartleben, Björn; Neves-Costa, Ana; Moita, Catarina; Pedroso, Dora; Pinto, Andreia; Marques, Sofia; Faridi, Hafeez; Costa, Paulo; Gozzelino, Raffaella; Zhao, Jimmy L; Soares, Miguel P; Gama-Carvalho, Margarida; Martinez, Jennifer; Zhang, Qingshuo; Döring, Gerd; Grompe, Markus; Simas, J Pedro; Huber, Tobias B; Baltimore, David; Gupta, Vineet; Green, Douglas R; Ferreira, João A; Moita, Luis F

    2013-11-14

    Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis. PMID:24184056

  4. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells

    PubMed Central

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-01-01

    Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague–Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo. PMID:26114388

  5. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells.

    PubMed

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-01-01

    Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague-Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo. PMID:26114388

  6. A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses

    PubMed Central

    Liang, Qin; Dexheimer, Thomas S; Zhang, Ping; Rosenthal, Andrew S; Villamil, Mark A; You, Changjun; Zhang, Qiuting; Chen, Junjun; Ott, Christine A; Sun, Hongmao; Luci, Diane K; Yuan, Bifeng; Simeonov, Anton; Jadhav, Ajit; Xiao, Hui; Wang, Yinsheng; Maloney, David J; Zhuang, Zhihao

    2014-01-01

    Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs. PMID:24531842

  7. Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

    PubMed Central

    Xiong, Jianhua; Todorova, Dilyana; Su, Ning-Yuan; Kim, Jinchul; Lee, Pei-Jen; Shen, Zhouxin; Briggs, Steven P.

    2015-01-01

    Mouse embryonic stem cells (ESCs) are genetically more stable than somatic cells, thereby preventing the passage of genomic abnormalities to their derivatives including germ cells. The underlying mechanisms, however, remain largely unclear. In this paper, we show that the stemness factor Sall4 is required for activating the critical Ataxia Telangiectasia Mutated (ATM)–dependent cellular responses to DNA double-stranded breaks (DSBs) in mouse ESCs and confer their resistance to DSB-induced cytotoxicity. Sall4 is rapidly mobilized to the sites of DSBs after DNA damage. Furthermore, Sall4 interacts with Rad50 and stabilizes the Mre11–Rad50–Nbs1 complex for the efficient recruitment and activation of ATM. Sall4 also interacts with Baf60a, a member of the SWI/SNF (switch/sucrose nonfermentable) ATP-dependent chromatin-remodeling complex, which is responsible for recruiting Sall4 to the site of DNA DSB damage. Our findings provide novel mechanisms to coordinate stemness of ESCs with DNA damage response, ensuring genomic stability during the expansion of ESCs. PMID:25733712

  8. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

    PubMed

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T; Passmore, Lori A; Patel, Ketan J; Olsen, Jesper V; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage. PMID:25557546

  9. BRIT1 regulates early DNA damage response, chromosomal integrity,and cancer

    SciTech Connect

    Rai, Rekha; Dai, Hui; Multani, Asha S.; Li, Kaiyi; Chin, Koei; Gray, Joe; Lahad, John P.; Liang, Jiyong; Mills, Gordon B.; Meric-Bernstam, Funda; Lin, Shiaw-Yih

    2006-08-24

    BRIT1, initially identified as an hTERT repressor, hasadditional functions at DNA damage checkpoints. Here, we demonstratedthat BRIT1 formed nuclear foci minutes after irradiation. The foci ofBRIT1 co-localized with 53BP1, MDC1, NBS1, ATM, RPA, and ATR. BRIT1 wasrequired for activation of these elements, indicating that BRIT1 is aproximal factor in the DNA damage response pathway. Depletion of BRIT1increased the accumulation of chromosomal aberrations. In addition,decreased levels of BRIT1 were detected in several types of human cancerwith BRIT1 expression being inversely correlated with genomic instabilityand metastasis. These results identify BRIT1 as a crucial DNA damageregulator in the ATM/ATR pathways and suggest that it functions as atumor suppressor gene.

  10. Microgravity increases DNA damage response in Caenorhabditis elegans during Shenzhou-8 spaceflight

    NASA Astrophysics Data System (ADS)

    Gao, Ying; Sun, Yeqing; Xu, Dan; Zhao, Lei; Xu, Jiamin

    DNA damage response (DDR) plays an important role in genome maintenance through cell cycle arrest followed by DNA repair and/or apoptosis. Perturbing DDR may elicit genomic instability, carcinogenesis, even cell death. Space radiation and microgravity both have been reported to cause DDR in mammal cells,while, in the space environment, the interaction of space radiation and microgravity on DDR is still controversial. To clarify the interaction, dauer larva of Caenorhabditis elegans were employed in Shenzhou-8 space mission and suffered space synthetic environment (RM) and space radiation (R) during 16.5-day spaceflight. mRNA microarray, qPCR and miRNA microarray were performed individually to detect the differences of transcriptome and microRNome affected by two environments. The results showed that, two fold genes were regulated more significantly by RM than by R. These regulated genes were involved in different physiological activities from each environment, which mainly involve in protein metabolic and modification processes in RM, and energy metabolic process in R. 21 of 500 DDR genes were extracted as significantly different expression in two space environments. DNA repair and apoptosis were enhanced by microgravity, since 18 of 21 genes were altered by RM specifically, including six “Response to DNA damage stimulus” genes, four “DNA repair” genes and eight “apoptosis process” genes. miRNAome also showed changes in response to microgravity. miRNA-81, 82, 124 and 795 were predicted to respond to RM and regulate DDR in C.elegans for the first time. These results suggest that microgravity increases the physiological activities to the space environment, especially enhance DNA damage response on transcription and post-transcriptional regulation in metazoan. We expect the finding provides new informations on synergetic effects between microgravity and radiation, and may be helpful for space risk assessment.

  11. Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970.

    PubMed

    Hall, Amy B; Newsome, Dave; Wang, Yuxin; Boucher, Diane M; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A; Milton, Sean; Murphy, Cheryl E; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M; Pollard, John R

    2014-07-30

    Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients. PMID:25010037

  12. Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970

    PubMed Central

    Boucher, Diane M.; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A.; Milton, Sean; Murphy, Cheryl E.; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M.; Pollard, John R.

    2014-01-01

    Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients. PMID:25010037

  13. Modulation of the DNA-damage response to HZE particles by shielding.

    PubMed

    Mukherjee, Bipasha; Camacho, Cristel Vanessa; Tomimatsu, Nozomi; Miller, Jack; Burma, Sandeep

    2008-10-01

    Ions of high atomic number and energy (HZE particles) pose a significant cancer risk to astronauts on prolonged space missions. On Earth, similar ions are being used for targeted cancer therapy. The properties of these particles can be drastically altered during passage through spacecraft shielding, therapy beam modulators, or the human body. Here, we have used pertinent responses to DNA double-strand breaks (DSBs) to understand the consequences of energy loss versus nuclear fragmentation of Fe ions during passage through shielding or tissue-equivalent materials. Phosphorylation of histone H2AX and recruitment of 53BP1 were used to generate 3D reconstructions of DNA damage in human cells and to follow its repair. Human cells are unable to repair a significant portion of DNA damage induced by Fe ions. DNA-PK and ATM are required, to different extents, for the partial repair of Fe-induced DNA damage. Aluminum shielding has little effect on DNA damage or its repair, confirming that the hulls of the Space Shuttle and the International Space Station afford scant protection against these particles. Lead shielding, on the other hand, exacerbates the effects of Fe ions due to energy loss during particle traversal. In sharp contrast, polyethylene (PE), a favored hydrogenous shield, results in DNA damage that is more amenable to repair presumably due to Fe-ion fragmentation. Human cells are indeed able to efficiently repair DSBs induced by chlorine ions and protons that represent fragmentation products of Fe. Interestingly, activation of the tumor suppressor p53 in Fe-irradiated cells is uniquely biphasic and culminates in the induction of high levels of p21 (Waf1/Cip1), p16 (INK4a) and senescence-associated beta-galactosidase activity. Surprisingly, these events occur even in the absence of ATM kinase implying that ATR may be a major responder to the complex DNA damage inflicted by Fe ions. Significantly, fragmentation of the Fe beam through PE attenuates these responses

  14. Mitochondrial DNA Damage and Diseases

    PubMed Central

    Singh, Gyanesh; Pachouri, U C; Khaidem, Devika Chanu; Kundu, Aman; Chopra, Chirag; Singh, Pushplata

    2015-01-01

    Various endogenous and environmental factors can cause mitochondrial DNA (mtDNA) damage.  One of the reasons for enhanced mtDNA damage could be its proximity to the source of oxidants, and lack of histone-like protective proteins. Moreover, mitochondria contain inadequate DNA repair pathways, and, diminished DNA repair capacity may be one of the factors responsible for high mutation frequency of the mtDNA. mtDNA damage might cause impaired mitochondrial function, and, unrepaired mtDNA damage has been frequently linked with several diseases. Exploration of mitochondrial perspective of diseases might lead to a better understanding of several diseases, and will certainly open new avenues for detection, cure, and prevention of ailments.

  15. Quantitative Proteomic Atlas of Ubiquitination and Acetylation in the DNA Damage Response

    PubMed Central

    Elia, Andrew E.H.; Boardman, Alexander P.; Wang, David C.; Huttlin, Edward L.; Everley, Robert A.; Dephoure, Noah; Zhou, Chunshui; Koren, Itay; Gygi, Steven P.; Elledge, Stephen J.

    2015-01-01

    Summary Execution of the DNA damage response (DDR) relies upon a dynamic array of protein modifications. Using quantitative proteomics, we have globally profiled ubiquitination, acetylation, and phosphorylation in response to ultraviolet and ionizing radiation. To improve acetylation site profiling, we developed the strategy FACET-IP. Our datasets of 33,500 ubiquitination and 16,740 acetylation sites provide valuable insight into DDR remodeling of the proteome. We find that K6- and K33-linked polyubiquitination undergo bulk increases in response to DNA damage, raising the possibility that these linkages are largely dedicated to DDR function. We also show that Cullin Ring Ligases mediate 10% of DNA damage induced ubiquitination events and that EXO1 is an SCF-Cyclin F substrate in the response to UV radiation. Our extensive datasets uncover additional regulated sites on known DDR players such as PCNA and identify previously unknown DDR targets such as CENPs, underscoring the broad impact of the DDR on cellular physiology. PMID:26051181

  16. DNA damage response in male gametes of Cyrtanthus mackenii during pollen tube growth

    PubMed Central

    Hirano, Tomonari; Takagi, Keiichi; Hoshino, Yoichiro; Abe, Tomoko

    2013-01-01

    Male gametophytes of plants are exposed to environmental stress and mutagenic agents during the double fertilization process and therefore need to repair the DNA damage in order to transmit the genomic information to the next generation. However, the DNA damage response in male gametes is still unclear. In the present study, we analysed the response to DNA damage in the generative cells of Cyrtanthus mackenii during pollen tube growth. A carbon ion beam, which can induce DNA double-strand breaks (DSBs), was used to irradiate the bicellular pollen, and then the irradiated pollen grains were cultured in a liquid culture medium. The male gametes were isolated from the cultured pollen tubes and used for immunofluorescence analysis. Although inhibitory effects on pollen tube growth were not observed after irradiation, sperm cell formation decreased significantly after high-dose irradiation. After high-dose irradiation, the cell cycle progression of generative cells was arrested at metaphase in pollen mitosis II, and phosphorylated H2AX (γH2AX) foci, an indicator of DSBs, were detected in the majority of the arrested cells. However, these foci were not detected in cells that were past metaphase. Cell cycle progression in irradiated generative cells is regulated by the spindle assembly checkpoint, and modification of the histones surrounding the DSBs was confirmed. These results indicate that during pollen tube growth generative cells can recognize and manage genomic lesions using DNA damage response pathways. In addition, the number of generative cells with γH2AX foci decreased with culture prolongation, suggesting that the DSBs in the generative cells are repaired. PMID:23550213

  17. Functions of Saccharomyces cerevisiae 14-3-3 proteins in response to DNA damage and to DNA replication stress.

    PubMed Central

    Lottersberger, Francisca; Rubert, Fabio; Baldo, Veronica; Lucchini, Giovanna; Longhese, Maria Pia

    2003-01-01

    Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes. We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage. In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles. Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases. Moreover, several bmh1 bmh2Delta mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polalpha-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response. Finally, the bmh1-169 bmh2Delta and bmh1-170 bmh2Delta mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability. PMID:14704161

  18. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells

    SciTech Connect

    Ahamed, Maqusood; Karns, Michael; Goodson, Michael; Rowe, John; Hussain, Saber M.; Schlager, John J.

    2008-12-15

    Silver nanoparticles (Ag NPs) have recently received much attention for their possible applications in biotechnology and life sciences. Ag NPs are of interest to defense and engineering programs for new material applications as well as for commercial purposes as an antimicrobial. However, little is known about the genotoxicity of Ag NPs following exposure to mammalian cells. This study was undertaken to examine the DNA damage response to polysaccharide surface functionalized (coated) and non-functionalized (uncoated) Ag NPs in two types of mammalian cells; mouse embryonic stem (mES) cells and mouse embryonic fibroblasts (MEF). Both types of Ag NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and phosphorylated-H2AX expression. Furthermore both of them induced cell death as measured by the annexin V protein expression and MTT assay. Our observations also suggested that the different surface chemistry of Ag NPs induce different DNA damage response: coated Ag NPs exhibited more severe damage than uncoated Ag NPs. The results suggest that polysaccharide coated particles are more individually distributed while agglomeration of the uncoated particles limits the surface area availability and access to membrane bound organelles.

  19. The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage

    PubMed Central

    Larsen, Dorthe H; Hari, Flurina; Clapperton, Julie A; Gwerder, Myriam; Gutsche, Katrin; Altmeyer, Matthias; Jungmichel, Stephanie; Toledo, Luis I; Fink, Daniel; Rask, Maj-Britt; Grøfte, Merete; Lukas, Claudia; Nielsen, Michael L; Smerdon, Stephen J; Lukas, Jiri; Stucki, Manuel

    2016-01-01

    Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in-trans signaling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identified TCOF1-Treacle, a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle-dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in-trans in the presence of distant chromosome breaks. PMID:25064736

  20. Modulation of ATR-mediated DNA damage checkpoint response by cryptochrome 1

    PubMed Central

    Kang, Tae-Hong; Leem, Sun-Hee

    2014-01-01

    Mammalian cryptochromes (Crys) are essential circadian clock factors implicated in diverse clock-independent physiological functions, including DNA damage responses. Here we show that Cry1 modulates the ATR-mediated DNA damage checkpoint (DDC) response by interacting with Timeless (Tim) in a time-of-day-dependent manner. The DDC capacity in response to UV irradiation showed a circadian rhythm. Interestingly, clock-deficient Cry1 and Cry2 double knockout (CryDKO) cells retained substantial DDC capacity compared with clock-proficient wild-type cells, although the Cry1-modulated oscillation of the DDC capacity was abolished in CryDKO cells. We found temporal interaction of Cry1 and Tim in the nucleus. When Cry1 was expressed in the nucleus, it was critical for circadian ATR activity. We regenerated rhythmic DDC responses by ectopically expressing Cry1 in CryDKO cells. In addition, we also investigated the DDC capacity in the liver of mice that were intraperitoneally injected with cisplatin at different circadian times (CT). When mice were injected at CT20, about 2-fold higher expression of phosphorylated minichromosome maintenance protein 2 (p-MCM2) was detected compared with mice injected at CT08, which consequently affected the removal rate of cisplatin-DNA adducts from genomic DNA. Taken together, our data demonstrate the intimate interaction between the circadian clock and the DDC system during genotoxic stress in clock-ticking cells. PMID:24489120

  1. Acute MUS81 depletion leads to replication fork slowing and a constitutive DNA damage response

    PubMed Central

    Xing, Meichun; Wang, Xiaohui; Palmai-Pallag, Timea; Shen, Huahao; Helleday, Thomas; Hickson, Ian D.; Ying, Songmin

    2015-01-01

    The MUS81 protein belongs to a conserved family of DNA structure-specific nucleases that play important roles in DNA replication and repair. Inactivation of the Mus81 gene in mice has no major deleterious consequences for embryonic development, although cancer susceptibility has been reported. We have investigated the role of MUS81 in human cells by acutely depleting the protein using shRNAs. We found that MUS81 depletion from human fibroblasts leads to accumulation of ssDNA and a constitutive DNA damage response that ultimately activates cellular senescence. Moreover, we show that MUS81 is required for efficient replication fork progression during an unperturbed S-phase, and for recovery of productive replication following replication stalling. These results demonstrate essential roles for the MUS81 nuclease in maintenance of replication fork integrity. PMID:26415217

  2. Aberrant DNA Damage Response Pathways May Predict the Outcome of Platinum Chemotherapy in Ovarian Cancer

    PubMed Central

    Stefanou, Dimitra T.; Bamias, Aristotelis; Episkopou, Hara; Kyrtopoulos, Soterios A.; Likka, Maria; Kalampokas, Theodore; Photiou, Stylianos; Gavalas, Nikos; Sfikakis, Petros P.; Dimopoulos, Meletios A.; Souliotis, Vassilis L.

    2015-01-01

    Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that

  3. Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

    PubMed Central

    Mitchell, Jonathan K.

    2012-01-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals. PMID:23035220

  4. Analysis of Individual Molecular Events of DNA Damage Response by Flow and Image Assisted Cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Skommer, Joanna; Wlodkowic, Donald

    2010-01-01

    This chapter describes molecular mechanisms of DNA damage response (DDR) and presents flow- and image-assisted cytometric approaches to assess these mechanisms and measure the extent of DDR in individual cells. DNA damage was induced by cell treatment with oxidizing agents, UV light, DNA topoisomerase I or II inhibitors, cisplatin, tobacco smoke, and by exogenous and endogenous oxidants. Chromatin relaxation (decondensation) is an early event of DDR chromatin that involves modification of high mobility group proteins (HMGs) and histone H1 and was detected by cytometry by analysis of the susceptibility of DNA in situ to denaturation using the metachromatic fluorochrome acridine orange. Translocation of the MRN complex consisting of Meiotic Recombination 11 Homolog A (Mre11), Rad50 homolog and Nijmegen Breakage Syndrome 1 (NMR1) into DNA damage sites was assessed by laser scanning cytometry as the increase in the intensity of maximal pixel as well as integral value of Mre11 immunofluorescence. Examples of cytometric detection of activation of Ataxia telangiectasia mutated (ATM), and Check 2 (Chk2) protein kinases using phospho-specific Abs targeting Ser1981 and Thr68 of these proteins, respectively are also presented. We also discuss approaches to correlate activation of ATM and Chk2 with phosphorylation of p53 on Ser15 and histone H2AX on Ser139 as well as with cell cycle position and DNA replication. The capability of laser scanning cytometry to quantify individual foci of phosphorylated H2AX and/or ATM that provides more dependable assessment of the presence of DNA double-strand breaks is outlined. The new microfluidic Lab-on-a-Chip platforms for interrogation of individual cells offer a novel approach for DDR cytometric analysis. PMID:21722802

  5. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

    PubMed Central

    Halaby, Marie-Jo; Li, Yan; Harris, Benjamin R.; Jiang, Shuxia; Miskimins, W. Keith; Cleary, Margot P.; Yang, Da-Qing

    2015-01-01

    Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) that is located at the 5′-untranslated region (UTR) of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80) has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA) following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress. PMID:26273641

  6. Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells

    PubMed Central

    Coluzzi, Elisa; Colamartino, Monica; Cozzi, Renata; Leone, Stefano; Meneghini, Carlo; O’Callaghan, Nathan; Sgura, Antonella

    2014-01-01

    One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect. PMID:25354277

  7. Development of DNA Damage Response Signaling Biomarkers using Automated, Quantitative Image Analysis

    PubMed Central

    Nikolaishvilli-Feinberg, Nana; Cohen, Stephanie M.; Midkiff, Bentley; Zhou, Yingchun; Olorvida, Mark; Ibrahim, Joseph G.; Omolo, Bernard; Shields, Janiel M.; Thomas, Nancy E.; Groben, Pamela A.; Kaufmann, William K.

    2014-01-01

    The DNA damage response (DDR) coordinates DNA repair with cell cycle checkpoints to ameliorate or mitigate the pathological effects of DNA damage. Automated quantitative analysis (AQUA) and Tissue Studio are commercial technologies that use digitized immunofluorescence microscopy images to quantify antigen expression in defined tissue compartments. Because DDR is commonly activated in cancer and may reflect genetic instability within the lesion, a method to quantify DDR in cancer offers potential diagnostic and/or prognostic value. In this study, both AQUA and Tissue Studio algorithms were used to quantify the DDR in radiation-damaged skin fibroblasts, melanoma cell lines, moles, and primary and metastatic melanomas. Digital image analysis results for three markers of DDR (γH2AX, P-ATM, P-Chk2) correlated with immunoblot data for irradiated fibroblasts, whereas only γH2AX and P-Chk2 correlated with immunoblot data in melanoma cell lines. Melanoma cell lines displayed substantial variation in γH2AX and P-Chk2 expression, and P-Chk2 expression was significantly correlated with radioresistance. Moles, primary melanomas, and melanoma metastases in brain, lung and liver displayed substantial variation in γH2AX expression, similar to that observed in melanoma cell lines. Automated digital analysis of immunofluorescent images stained for DDR biomarkers may be useful for predicting tumor response to radiation and chemotherapy. PMID:24309508

  8. The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

    PubMed

    Danilova, Nadia; Bibikova, Elena; Covey, Todd M; Nathanson, David; Dimitrova, Elizabeth; Konto, Yoan; Lindgren, Anne; Glader, Bertil; Radu, Caius G; Sakamoto, Kathleen M; Lin, Shuo

    2014-07-01

    Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA. PMID:24812435

  9. The ribosomal protein S26 regulates p53 activity in response to DNA damage.

    PubMed

    Cui, D; Li, L; Lou, H; Sun, H; Ngai, S-M; Shao, G; Tang, J

    2014-04-24

    Ribosomal proteins have emerged as novel regulators of the Mdm2-p53 feedback loop, especially in the context of ribosomal stress. RPS26 is a recently identified Diamond-Blackfan Anemia-related ribosomal protein and its role in p53 activation has not been previously explored. In this study we found knockdown of RPS26 induced p53 stabilization and activation via a RPL11-dependent mechanism, resulting in p53-dependent cell growth inhibition. Moreover, RPS26 has the ability to interact with Mdm2 and inhibits Mdm2-mediated p53 ubiquitination that leads to p53 stabilization upon overexpression. Importantly, we discovered that RPS26 knockdown impaired p53's ability to transcriptionally activate its target genes in response to DNA damage, without affecting its stability. Accordingly, the cells lost the ability to induce G2/M cell cycle arrest. We further found that upon RPS26 knockdown, the DNA damage induced recruitment of p53 to the promoters of its target genes and p53 acetylation were both greatly reduced. In addition, RPS26 can interact with p53 independent of Mdm2 and coexist in a complex with p53 and p300. These data establish a role of RPS26 in DNA damage response by directly influencing p53 transcriptional activity, and suggest that RPS26 acts distinctively in different scenarios of p53 activation. Our finding also implicates p53 transcriptional activity control as an important mechanism of p53 regulation by ribosomal proteins. PMID:23728348

  10. The tumour suppressor CYLD regulates the p53 DNA damage response

    PubMed Central

    Fernández-Majada, Vanesa; Welz, Patrick-Simon; Ermolaeva, Maria A.; Schell, Michael; Adam, Alexander; Dietlein, Felix; Komander, David; Büttner, Reinhard; Thomas, Roman K.; Schumacher, Björn; Pasparakis, Manolis

    2016-01-01

    The tumour suppressor CYLD is a deubiquitinase previously shown to inhibit NF-κB, MAP kinase and Wnt signalling. However, the tumour suppressing mechanisms of CYLD remain poorly understood. Here we show that loss of CYLD catalytic activity causes impaired DNA damage-induced p53 stabilization and activation in epithelial cells and sensitizes mice to chemical carcinogen-induced intestinal and skin tumorigenesis. Mechanistically, CYLD interacts with and deubiquitinates p53 facilitating its stabilization in response to genotoxic stress. Ubiquitin chain-restriction analysis provides evidence that CYLD removes K48 ubiquitin chains from p53 indirectly by cleaving K63 linkages, suggesting that p53 is decorated with complex K48/K63 chains. Moreover, CYLD deficiency also diminishes CEP-1/p53-dependent DNA damage-induced germ cell apoptosis in the nematode Caenorhabditis elegans. Collectively, our results identify CYLD as a deubiquitinase facilitating DNA damage-induced p53 activation and suggest that regulation of p53 responses to genotoxic stress contributes to the tumour suppressor function of CYLD. PMID:27561390

  11. The tumour suppressor CYLD regulates the p53 DNA damage response.

    PubMed

    Fernández-Majada, Vanesa; Welz, Patrick-Simon; Ermolaeva, Maria A; Schell, Michael; Adam, Alexander; Dietlein, Felix; Komander, David; Büttner, Reinhard; Thomas, Roman K; Schumacher, Björn; Pasparakis, Manolis

    2016-01-01

    The tumour suppressor CYLD is a deubiquitinase previously shown to inhibit NF-κB, MAP kinase and Wnt signalling. However, the tumour suppressing mechanisms of CYLD remain poorly understood. Here we show that loss of CYLD catalytic activity causes impaired DNA damage-induced p53 stabilization and activation in epithelial cells and sensitizes mice to chemical carcinogen-induced intestinal and skin tumorigenesis. Mechanistically, CYLD interacts with and deubiquitinates p53 facilitating its stabilization in response to genotoxic stress. Ubiquitin chain-restriction analysis provides evidence that CYLD removes K48 ubiquitin chains from p53 indirectly by cleaving K63 linkages, suggesting that p53 is decorated with complex K48/K63 chains. Moreover, CYLD deficiency also diminishes CEP-1/p53-dependent DNA damage-induced germ cell apoptosis in the nematode Caenorhabditis elegans. Collectively, our results identify CYLD as a deubiquitinase facilitating DNA damage-induced p53 activation and suggest that regulation of p53 responses to genotoxic stress contributes to the tumour suppressor function of CYLD. PMID:27561390

  12. Development of DNA damage response signaling biomarkers using automated, quantitative image analysis.

    PubMed

    Nikolaishvilli-Feinberg, Nana; Cohen, Stephanie M; Midkiff, Bentley; Zhou, Yingchun; Olorvida, Mark; Ibrahim, Joseph G; Omolo, Bernard; Shields, Janiel M; Thomas, Nancy E; Groben, Pamela A; Kaufmann, William K; Miller, C Ryan

    2014-03-01

    The DNA damage response (DDR) coordinates DNA repair with cell cycle checkpoints to ameliorate or mitigate the pathological effects of DNA damage. Automated quantitative analysis (AQUA) and Tissue Studio are commercial technologies that use digitized immunofluorescence microscopy images to quantify antigen expression in defined tissue compartments. Because DDR is commonly activated in cancer and may reflect genetic instability within the lesion, a method to quantify DDR in cancer offers potential diagnostic and/or prognostic value. In this study, both AQUA and Tissue Studio algorithms were used to quantify the DDR in radiation-damaged skin fibroblasts, melanoma cell lines, moles, and primary and metastatic melanomas. Digital image analysis results for three markers of DDR (γH2AX, P-ATM, P-Chk2) correlated with immunoblot data for irradiated fibroblasts, whereas only γH2AX and P-Chk2 correlated with immunoblot data in melanoma cell lines. Melanoma cell lines displayed substantial variation in γH2AX and P-Chk2 expression, and P-Chk2 expression was significantly correlated with radioresistance. Moles, primary melanomas, and melanoma metastases in brain, lung and liver displayed substantial variation in γH2AX expression, similar to that observed in melanoma cell lines. Automated digital analysis of immunofluorescent images stained for DDR biomarkers may be useful for predicting tumor response to radiation and chemotherapy. PMID:24309508

  13. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    PubMed Central

    Fox, Jennifer T.; Sakamuru, Srilatha; Huang, Ruili; Teneva, Nedelina; Simmons, Steven O.; Xia, Menghang; Tice, Raymond R.; Austin, Christopher P.; Myung, Kyungjae

    2012-01-01

    Human ATAD5 is a biomarker for identifying genotoxic compounds because ATAD5 protein levels increase posttranscriptionally in response to DNA damage. We screened over 4,000 compounds with a cell-based quantitative high-throughput ATAD5-luciferase assay detecting genotoxic compounds. We identified 22 antioxidants, including resveratrol, genistein, and baicalein, that are currently used or investigated for the treatment of cardiovascular disease, type 2 diabetes, osteopenia, osteoporosis, and chronic hepatitis, as well as for antiaging. Treatment of dividing cells with these compounds induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anticancer drugs. Furthermore, resveratrol and genistein killed multidrug-resistant cancer cells. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents. PMID:22431602

  14. SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage.

    PubMed

    Yin, Yili; Seifert, Anne; Chua, Joy Shijia; Maure, Jean-François; Golebiowski, Filip; Hay, Ronald T

    2012-06-01

    Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress. PMID:22661230

  15. DNA damage response and prostate cancer: defects, regulation and therapeutic implications

    PubMed Central

    Karanika, Styliani; Karantanos, Theodoros; Li, Likun; Corn, Paul G.; Thompson, Timothy C.

    2014-01-01

    DNA damage response (DDR) includes the activation of numerous cellular activities that prevent duplication of DNA lesions and maintain genomic integrity, which is critical for the survival of normal and cancer cells. Specific genes involved in the DDR such as BRCA1/2 and P53 are mutated during prostate cancer progression, while various oncogenic signaling such as Akt and c-Myc are activated, enhancing the replication stress and increasing the genomic instability of cancer cells. These events may render prostate cancer cells particularly sensitive to inhibition of specific DDR pathways, such as PARP in homologous recombination (HR) DNA repair and Chk1 in cell cycle checkpoint and DNA repair, creating opportunities for synthetic lethality or synergistic cytotoxicity. Recent reports highlight the critical role of androgen receptor (AR) as a regulator of DDR genes, providing a rationale for combining DNA-damaging agents or targeted DDR inhibitors with hormonal manipulation or AR inhibition as treatment for aggressive disease. The aims of this review are to discuss specific DDR defects in prostate cancer that occur during disease progression, to summarize recent advances in understanding the regulation of DDR in prostate cancer, and to present potential therapeutic opportunities through combinational targeting of the intact components of DDR signaling pathways. PMID:25132269

  16. DNA damage response and prostate cancer: defects, regulation and therapeutic implications.

    PubMed

    Karanika, S; Karantanos, T; Li, L; Corn, P G; Thompson, T C

    2015-05-28

    DNA damage response (DDR) includes the activation of numerous cellular activities that prevent duplication of DNA lesions and maintain genomic integrity, which is critical for the survival of normal and cancer cells. Specific genes involved in the DDR such as BRCA1/2 and P53 are mutated during prostate cancer progression, while various oncogenic signaling such as Akt and c-Myc are activated, enhancing the replication stress and increasing the genomic instability of cancer cells. These events may render prostate cancer cells particularly sensitive to inhibition of specific DDR pathways, such as PARP in homologous recombination DNA repair and Chk1 in cell cycle checkpoint and DNA repair, creating opportunities for synthetic lethality or synergistic cytotoxicity. Recent reports highlight the critical role of androgen receptor (AR) as a regulator of DDR genes, providing a rationale for combining DNA-damaging agents or targeted DDR inhibitors with hormonal manipulation or AR inhibition as treatment for aggressive disease. The aims of this review are to discuss specific DDR defects in prostate cancer that occur during disease progression, to summarize recent advances in understanding the regulation of DDR in prostate cancer, and to present potential therapeutic opportunities through combinational targeting of the intact components of DDR signaling pathways. PMID:25132269

  17. A novel transcription factor gene FHS1 is involved in the DNA damage response in Fusarium graminearum

    PubMed Central

    Son, Hokyoung; Fu, Minmin; Lee, Yoonji; Lim, Jae Yun; Min, Kyunghun; Kim, Jin-Cheol; Choi, Gyung Ja; Lee, Yin-Won

    2016-01-01

    Cell cycle regulation and the maintenance of genome integrity are crucial for the development and virulence of the pathogenic plant fungus Fusarium graminearum. To identify transcription factors (TFs) related to these processes, four DNA-damaging agents were applied to screen a F. graminearum TF mutant library. Sixteen TFs were identified to be likely involved in DNA damage responses. Fhs1 is a fungal specific Zn(II)2Cys6 TF that localises exclusively to nuclei. fhs1 deletion mutants were hypersensitive to hydroxyurea and defective in mitotic cell division. Moreover, deletion of FHS1 resulted in defects in perithecia production and virulence and led to the accumulation of DNA damage. Our genetic evidence demonstrated that the FHS1-associated signalling pathway for DNA damage response is independent of the ATM or ATR pathways. This study identified sixteen genes involved in the DNA damage response and is the first to characterise the novel transcription factor gene FHS1, which is involved in the DNA damage response. The results provide new insights into mechanisms underlying DNA damage responses in fungi, including F. graminearum. PMID:26888604

  18. A novel transcription factor gene FHS1 is involved in the DNA damage response in Fusarium graminearum.

    PubMed

    Son, Hokyoung; Fu, Minmin; Lee, Yoonji; Lim, Jae Yun; Min, Kyunghun; Kim, Jin-Cheol; Choi, Gyung Ja; Lee, Yin-Won

    2016-01-01

    Cell cycle regulation and the maintenance of genome integrity are crucial for the development and virulence of the pathogenic plant fungus Fusarium graminearum. To identify transcription factors (TFs) related to these processes, four DNA-damaging agents were applied to screen a F. graminearum TF mutant library. Sixteen TFs were identified to be likely involved in DNA damage responses. Fhs1 is a fungal specific Zn(II)2Cys6 TF that localises exclusively to nuclei. fhs1 deletion mutants were hypersensitive to hydroxyurea and defective in mitotic cell division. Moreover, deletion of FHS1 resulted in defects in perithecia production and virulence and led to the accumulation of DNA damage. Our genetic evidence demonstrated that the FHS1-associated signalling pathway for DNA damage response is independent of the ATM or ATR pathways. This study identified sixteen genes involved in the DNA damage response and is the first to characterise the novel transcription factor gene FHS1, which is involved in the DNA damage response. The results provide new insights into mechanisms underlying DNA damage responses in fungi, including F. graminearum. PMID:26888604

  19. DNA damage response (DDR) via NKX3.1 expression in prostate cells.

    PubMed

    Erbaykent-Tepedelen, Burcu; Karamil, Selda; Gonen-Korkmaz, Ceren; Korkmaz, Kemal S

    2014-05-01

    It has been reported that NKX3.1 an androgen-regulated homeobox gene restricted to prostate and testicular tissues, encodes a homeobox protein, which transcriptionally regulates oxidative damage responses and enhances topoisomerase I re-ligation by a direct interaction with the ATM protein in prostate cells. In this study, we aimed to investigate the role of NKX3.1 in DNA double-strand break (DSB) repair. We demonstrate that the DNA damage induced by CPT-11 (irinotecan, a topo I inhibitor), doxorubicin (a topo II inhibitor), and H2O2 (a mediator of oxidative damage), but not by etoposide (another topo II inhibitor), is negatively influenced by NKX3.1 expression. We also examined γH2AX((S139)) foci formation and observed that the overexpression of NKX3.1 resulted a remarkable decrease in the formation of γH2AX((S139)) foci. Intriguingly, we observed in NKX3.1 silencing studies that the depletion of NKX3.1 correlated with a significant decrease in the levels of p-ATM((S1981)) and γH2AX((S139)). The data imply that the DNA damage response (DDR) can be altered, perhaps via a decrease in the topoisomerase I re-ligation function; this is consistent with the physical association of NKX3.1 with DDR mediators upon treatment of both PC-3 and LNCaP cells with CPT-11. Furthermore, the depletion of NKX3.1 resulted in a G1/S progression via the facilitation of an increase in E2F stabilization concurrent with the suppressed DDR. Thus, the topoisomerase I inhibitor-mediated DNA damage enhanced the physical association of NKX3.1 with γH2AX((S139)) on the chromatin in LNCaP cells, whereas NKX3.1 in the soluble fraction was associated with p-ATM((S1981)) and RAD50 in these cells. Overall, the data suggest that androgens and NKX3.1 expression regulate the progression of the cell cycle and concurrently activate the DDR. Therefore, androgen withdrawal may augment the development of an error-prone phenotype and, subsequently, the loss of DNA damage control during prostate cancer

  20. Replicating Damaged DNA in Eukaryotes

    PubMed Central

    Chatterjee, Nimrat; Siede, Wolfram

    2013-01-01

    DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail. PMID:24296172

  1. DNA Damage Response in Neonatal and Adult Stromal Cells Compared With Induced Pluripotent Stem Cells

    PubMed Central

    Liedtke, Stefanie; Biebernick, Sophie; Radke, Teja Falk; Stapelkamp, Daniela; Coenen, Carolin; Zaehres, Holm; Fritz, Gerhard; Kogler, Gesine

    2015-01-01

    Comprehensive analyses comparing individual DNA damage response (DDR) of induced pluripotent stem cells (iPSCs) with neonatal stromal cells with respect to their developmental age are limited. The imperative necessity of providing developmental age-matched cell sources for meaningful toxicological drug safety assessments in replacement of animal-based testing strategies is evident. Here, DDR after radiation or treatment with N-methyl-N-nitrosurea (MNU) was determined in iPSCs compared with neonatal and bone marrow stromal cells. Neonatal and adult stromal cells showed no significant morphologically detectable cytotoxicity following treatment with 1 Gy or 1 mM MNU, whereas iPSCs revealed a much higher sensitivity. Foci analyses revealed an effective DNA repair in stromal cell types and iPSCs, as reflected by a rapid formation and disappearance of phosphorylated ATM and γH2AX foci. Furthermore, quantitative polymerase chain reaction analyses revealed the highest basic expression level of DDR and repair-associated genes in iPSCs, followed by neonatal stromal cells and adult stromal cells with the lowest expression levels. In addition, the influence of genotoxic stress prior to and during osteogenic differentiation of neonatal and adult stromal cells was analyzed applying common differentiation procedures. Experiments presented here suggest a developmental age-dependent basic expression level of genes involved in the processing of DNA damage. In addition a differentiation-dependent downregulation of repair genes was observed during osteogenesis. These results strongly support the requirement to provide adequate cell sources for toxicological in vitro drug testing strategies that match to the developmental age and differentiation status of the presumptive target cell of interest. Significance The results obtained in this study advance the understanding of DNA damage processing in human neonatal stromal cells as compared with adult stromal cells and induced pluripotent

  2. Trm9 Catalyzed tRNA Modifications Link Translation to the DNA Damage Response

    PubMed Central

    Begley, Ulrike; Dyavaiah, Madhu; Patil, Ashish; Rooney, John P.; DiRenzo, Dan; Young, Christine M.; Conklin, Douglas S.; Zitomer, Richard S.; Begley, Thomas J.

    2007-01-01

    Summary Transcriptional and post-translational signals are known mechanisms which promote efficient responses to DNA damage. We have identified Saccharomyces cerevisiae tRNA methyltransferase 9 (Trm9) as an enzyme that prevents cell death via translational enhancement of DNA damage response proteins. Trm9 methylates the uridine wobble base of tRNAARG(UCU) and tRNAGLU(UUC). We used computational and molecular approaches to predict that Trm9 enhances the translation of some transcripts over-represented with specific arginine and glutamic acid codons. We found that translation elongation factor 3 (YEF3) and the ribonucleotide reductase (RNR1 and RNR3) large subunits are over-represented with specific arginine and glutamic acid codons, and demonstrated that Trm9 significantly enhances Yef3, Rnr1, and Rnr3 protein levels. Additionally, we identified 425 genes, which included YEF3, RNR1, and RNR3, with a unique codon usage pattern linked to Trm9. We propose that Trm9-specific tRNA modifications enhance codon-specific translation elongation and promote increased levels of key damage response proteins. PMID:18082610

  3. Stop pulling my strings - what telomeres taught us about the DNA damage response.

    PubMed

    Lazzerini-Denchi, Eros; Sfeir, Agnel

    2016-06-01

    Mammalian cells have evolved specialized mechanisms to sense and repair double-strand breaks (DSBs) to maintain genomic stability. However, in certain cases, the activity of these pathways can lead to aberrant DNA repair, genomic instability and tumorigenesis. One such case is DNA repair at the natural ends of linear chromosomes, known as telomeres, which can lead to chromosome-end fusions. Here, we review data obtained over the past decade and discuss the mechanisms that protect mammalian chromosome ends from the DNA damage response. We also discuss how telomere research has helped to uncover key steps in DSB repair. Last, we summarize how dysfunctional telomeres and the ensuing genomic instability drive the progression of cancer. PMID:27165790

  4. RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response.

    PubMed

    Groocock, Lynda M; Nie, Minghua; Prudden, John; Moiani, Davide; Wang, Tao; Cheltsov, Anton; Rambo, Robert P; Arvai, Andrew S; Hitomi, Chiharu; Tainer, John A; Luger, Karolin; Perry, J Jefferson P; Lazzerini-Denchi, Eros; Boddy, Michael N

    2014-05-01

    The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining. PMID:24714598

  5. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-Induced DNA Damages in Confluent Human Fibroblasts

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2016-01-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 µg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by foci and pattern counting of phosphorylated histone protein H2AX (?-H2AX). The cells on the ISS showed modestly increased average foci counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profile of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in ?-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not significantly affect

  6. DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis.

    PubMed

    Oya, Elisabeth; Ovrevik, Johan; Arlt, Volker M; Nagy, Eszter; Phillips, David H; Holme, Jørn A

    2011-11-01

    Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound. PMID:21715570

  7. Ccr4-not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae.

    PubMed

    Traven, Ana; Hammet, Andrew; Tenis, Nora; Denis, Clyde L; Heierhorst, Jörg

    2005-01-01

    DNA damage checkpoints regulate gene expression at the transcriptional and post-transcriptional level. Some components of the yeast Ccr4-Not complex, which regulates transcription as well as transcript turnover, have previously been linked to DNA damage responses, but it is unclear if this involves transcriptional or post-transcriptional functions. Here we show that CCR4 and CAF1, which together encode the major cytoplasmic mRNA deadenylase complex, have complex genetic interactions with the checkpoint genes DUN1, MRC1, RAD9, and RAD17 in response to DNA-damaging agents hydroxyurea (HU) and methylmethane sulfonate (MMS). The exonuclease-inactivating ccr4-1 point mutation mimics ccr4Delta phenotypes, including synthetic HU hypersensitivity with dun1Delta, demonstrating that Ccr4-Not mRNA deadenylase activity is required for DNA damage responses. However, ccr4Delta and caf1Delta DNA damage phenotypes and genetic interactions with checkpoint genes are not identical, and deletions of some Not components that are believed to predominantly function at the transcriptional level rather than mRNA turnover, e.g., not5Delta, also lead to increased DNA damage sensitivity and synthetic HU hypersensitivity with dun1Delta. Taken together, our data thus suggest that both transcriptional and post-transcriptional functions of the Ccr4-Not complex contribute to the DNA damage response affecting gene expression in a complex manner. PMID:15466434

  8. Ccr4-Not Complex mRNA Deadenylase Activity Contributes to DNA Damage Responses in Saccharomyces cerevisiae

    PubMed Central

    Traven, Ana; Hammet, Andrew; Tenis, Nora; Denis, Clyde L.; Heierhorst, Jörg

    2005-01-01

    DNA damage checkpoints regulate gene expression at the transcriptional and post-transcriptional level. Some components of the yeast Ccr4-Not complex, which regulates transcription as well as transcript turnover, have previously been linked to DNA damage responses, but it is unclear if this involves transcriptional or post-transcriptional functions. Here we show that CCR4 and CAF1, which together encode the major cytoplasmic mRNA deadenylase complex, have complex genetic interactions with the checkpoint genes DUN1, MRC1, RAD9, and RAD17 in response to DNA-damaging agents hydroxyurea (HU) and methylmethane sulfonate (MMS). The exonuclease-inactivating ccr4-1 point mutation mimics ccr4Δ phenotypes, including synthetic HU hypersensitivity with dun1Δ, demonstrating that Ccr4-Not mRNA deadenylase activity is required for DNA damage responses. However, ccr4Δ and caf1Δ DNA damage phenotypes and genetic interactions with checkpoint genes are not identical, and deletions of some Not components that are believed to predominantly function at the transcriptional level rather than mRNA turnover, e.g., not5Δ, also lead to increased DNA damage sensitivity and synthetic HU hypersensitivity with dun1Δ. Taken together, our data thus suggest that both transcriptional and post-transcriptional functions of the Ccr4-Not complex contribute to the DNA damage response affecting gene expression in a complex manner. PMID:15466434

  9. FBXL5-mediated degradation of single-stranded DNA-binding protein hSSB1 controls DNA damage response

    PubMed Central

    Chen, Zhi-Wei; Liu, Bin; Tang, Nai-Wang; Xu, Yun-Hua; Ye, Xiang-Yun; Li, Zi-Ming; Niu, Xiao-Min; Shen, Sheng-Ping; Lu, Shun; Xu, Ling

    2014-01-01

    Human single-strand (ss) DNA binding proteins 1 (hSSB1) has been shown to participate in DNA damage response and maintenance of genome stability by regulating the initiation of ATM-dependent signaling. ATM phosphorylates hSSB1 and prevents hSSB1 from ubiquitin-proteasome-mediated degradation. However, the E3 ligase that targets hSSB1 for destruction is still unknown. Here, we report that hSSB1 is the bona fide substrate for an Fbxl5-containing SCF (Skp1-Cul1-F box) E3 ligase. Fbxl5 interacts with and targets hSSB1 for ubiquitination and degradation, which could be prevented by ATM-mediated hSSB1 T117 phosphorylation. Furthermore, cells overexpression of Fbxl5 abrogated the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets and exhibited increased radiosensitivity, chemosensitivity and defective checkpoint activation after genotoxic stress stimuli. Moreover, the protein levels of hSSB1 and Fbxl5 showed an inverse correlation in lung cancer cells lines and clinical lung cancer samples. Therefore, Fbxl5 may negatively modulate hSSB1 to regulate DNA damage response, implicating Fbxl5 as a novel, promising therapeutic target for lung cancers. PMID:25249620

  10. FBXL5-mediated degradation of single-stranded DNA-binding protein hSSB1 controls DNA damage response.

    PubMed

    Chen, Zhi-Wei; Liu, Bin; Tang, Nai-Wang; Xu, Yun-Hua; Ye, Xiang-Yun; Li, Zi-Ming; Niu, Xiao-Min; Shen, Sheng-Ping; Lu, Shun; Xu, Ling

    2014-10-01

    Human single-strand (ss) DNA binding proteins 1 (hSSB1) has been shown to participate in DNA damage response and maintenance of genome stability by regulating the initiation of ATM-dependent signaling. ATM phosphorylates hSSB1 and prevents hSSB1 from ubiquitin-proteasome-mediated degradation. However, the E3 ligase that targets hSSB1 for destruction is still unknown. Here, we report that hSSB1 is the bona fide substrate for an Fbxl5-containing SCF (Skp1-Cul1-F box) E3 ligase. Fbxl5 interacts with and targets hSSB1 for ubiquitination and degradation, which could be prevented by ATM-mediated hSSB1 T117 phosphorylation. Furthermore, cells overexpression of Fbxl5 abrogated the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets and exhibited increased radiosensitivity, chemosensitivity and defective checkpoint activation after genotoxic stress stimuli. Moreover, the protein levels of hSSB1 and Fbxl5 showed an inverse correlation in lung cancer cells lines and clinical lung cancer samples. Therefore, Fbxl5 may negatively modulate hSSB1 to regulate DNA damage response, implicating Fbxl5 as a novel, promising therapeutic target for lung cancers. PMID:25249620

  11. ABRO1 suppresses tumourigenesis and regulates the DNA damage response by stabilizing p53

    PubMed Central

    Zhang, Jianhong; Cao, Mengmeng; Dong, Jiahong; Li, Changyan; Xu, Wangxiang; Zhan, Yiqun; Wang, Xiaohui; Yu, Miao; Ge, Changhui; Ge, Zhiqiang; Yang, Xiaoming

    2014-01-01

    Abraxas brother 1 (ABRO1) has been reported to be a component of the BRISC complex, a multiprotein complex that specifically cleaves ‘Lys-63’-linked ubiquitin. However, current knowledge of the functions of ABRO1 is limited. Here we report that ABRO1 is frequently downregulated in human liver, kidney, breast and thyroid gland tumour tissues. Depletion of ABRO1 in cancer cells reduces p53 levels and enhances clone formation and cellular transformation. Conversely, overexpression of ABRO1 suppresses cell proliferation and tumour formation in a p53-dependent manner. We further show that ABRO1 stabilizes p53 by facilitating the interaction of p53 with USP7. DNA-damage induced accumulation of endogenous ABRO1 as well as translocation of ABRO1 to the nucleus, and the induction of p53 by DNA damage is almost completely attenuated by ABRO1 depletion. Our study shows that ABRO1 is a novel p53 regulator that plays an important role in tumour suppression and the DNA damage response. PMID:25283148

  12. RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response

    PubMed Central

    Poulsen, Sara L.; Hansen, Rebecca K.; Wagner, Sebastian A.; van Cuijk, Loes; van Belle, Gijsbert J.; Streicher, Werner; Wikström, Mats; Choudhary, Chunaram; Houtsmuller, Adriaan B.; Marteijn, Jurgen A.; Bekker-Jensen, Simon

    2013-01-01

    Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13–Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response. PMID:23751493

  13. RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response.

    PubMed

    Poulsen, Sara L; Hansen, Rebecca K; Wagner, Sebastian A; van Cuijk, Loes; van Belle, Gijsbert J; Streicher, Werner; Wikström, Mats; Choudhary, Chunaram; Houtsmuller, Adriaan B; Marteijn, Jurgen A; Bekker-Jensen, Simon; Mailand, Niels

    2013-06-10

    Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13-Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response. PMID:23751493

  14. Is post-transcriptional stabilization, splicing and translation of selective mRNAs a key to the DNA damage response?

    PubMed Central

    2011-01-01

    In response to DNA damage, cells activate a complex, kinase-based signaling network that consists of two components—a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes to arrest the cell cycle along with recruitment of repair machinery to damaged DNA, followed by a delayed transcriptional response that promotes cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves post-transcriptional control of mRNA stability, splicing and translation as a critical part of the DNA damage response. Here, we describe recent work implicating DNA damage-dependent modification of RNA-binding proteins that are responsible for some of these mRNA effects, highlighting recent work on post-transcriptional regulation of the cell cycle checkpoint protein/apoptosis inducer Gadd45α by the checkpoint kinase MAPKAP Kinase-2. PMID:21173571

  15. Adaptive Response in Mice Exposed to 900 MHz Radiofrequency Fields: Primary DNA Damage

    PubMed Central

    Zhou, Zhen; Zhang, Jie; Tong, Jian; Cao, Yi

    2012-01-01

    The phenomenon of adaptive response (AR) in animal and human cells exposed to ionizing radiation is well documented in scientific literature. We have examined whether such AR could be induced in mice exposed to non-ionizing radiofrequency fields (RF) used for wireless communications. Mice were pre-exposed to 900 MHz RF at 120 µW/cm2 power density for 4 hours/day for 1, 3, 5, 7 and 14 days and then subjected to an acute dose of 3 Gy γ-radiation. The primary DNA damage in the form of alkali labile base damage and single strand breaks in the DNA of peripheral blood leukocytes was determined using the alkaline comet assay. The results indicated that the extent of damage in mice which were pre-exposed to RF for 1 day and then subjected to γ-radiation was similar and not significantly different from those exposed to γ-radiation alone. However, mice which were pre-exposed to RF for 3, 5, 7 and 14 days showed progressively decreased damage and was significantly different from those exposed to γ-radiation alone. Thus, the data indicated that RF pre-exposure is capable of inducing AR and suggested that the pre-exposure for more than 4 hours for 1 day is necessary to elicit such AR. PMID:22389679

  16. Role of DNA damage response pathways in preventing carcinogenesis caused by intrinsic replication stress.

    PubMed

    Wallace, M D; Southard, T L; Schimenti, K J; Schimenti, J C

    2014-07-10

    Defective DNA replication can result in genomic instability, cancer and developmental defects. To understand the roles of DNA damage response (DDR) genes on carcinogenesis in mutants defective for core DNA replication components, we utilized the Mcm4(Chaos3/Chaos3) ('Chaos3') mouse model that, by virtue of an amino-acid alteration in MCM4 that destabilizes the MCM2-7 DNA replicative helicase, has fewer dormant replication origins and an increased number of stalled replication forks. This leads to genomic instability and cancer in most Chaos3 mice. We found that animals doubly mutant for Chaos3 and components of the ataxia telangiectasia-mutated (ATM) double-strand break response pathway (Atm, p21/Cdkn1a and Chk2/Chek2) had decreased tumor latency and/or increased tumor susceptibility. Tumor latency and susceptibility differed between genetic backgrounds and genders, with females demonstrating an overall greater cancer susceptibility to Atm and p21 deficiency than males. Atm deficiency was semilethal in the Chaos3 background and impaired embryonic fibroblast proliferation, suggesting that ATM drug inhibitors might be useful against tumors with DNA replication defects. Hypomorphism for the 9-1-1 component Hus1 did not affect tumor latency or susceptibility in Chaos3 animals, and tumors in these mice did not exhibit impaired ATR pathway signaling. These and other data indicate that under conditions of systemic replication stress, the ATM pathway is particularly important both for cancer suppression and viability during development. PMID:23975433

  17. Differential requirements for H/ACA ribonucleoprotein components in cell proliferation and response to DNA damage.

    PubMed

    Lin, Ping; Mobasher, Maral E; Hakakian, Yasaman; Kakarla, Veena; Naseem, Anum F; Ziai, Heliya; Alawi, Faizan

    2015-12-01

    H/ACA ribonucleoproteins (RNPs) are comprised of four conserved proteins, dyskerin, NHP2, NOP10, and GAR1, and a function-specifying, noncoding H/ACA RNA. H/ACA RNPs contribute to telomerase assembly and stabilization, and posttranscriptional processing of nascent ribosomal RNA and spliceosomal RNA. However, very little is known about the coordinated action of the four proteins in other biologic processes. As described herein, we observed a differential requirement for the proteins in cell proliferation and identified a possible reliance for these factors in regulation of specific DNA damage biomarkers. In particular, GAR1 expression was upregulated following exposure to all forms of genotoxic stress tested. In contrast, levels of the other proteins were either reduced or unaffected. Only GAR1 showed an altered subcellular localization with a shift from the nucleolus to the nucleoplasm after ultraviolet-C irradiation and doxorubicin treatments. Transient siRNA-mediated depletion of GAR1 and dyskerin arrested cell proliferation, whereas loss of either NHP2 or NOP10 had no effect. Finally, loss of dyskerin, GAR1, NHP2, and NOP10, respectively, limited the accumulation of DNA damage biomarkers. However, the individual responses were dependent upon the specific type of damage incurred. In general, loss of GAR1 had the most suppressive effect on the biomarkers tested. Since the specific responses to genotoxic stress, the contribution of each protein to cell proliferation, and the activation of DNA damage biomarkers were not equivalent, this suggests the possibility that at least some of the proteins, most notably GAR1, may potentially function independently of their respective roles within H/ACA RNP complexes. PMID:26265134

  18. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

    SciTech Connect

    Yoshimura, Akari; Kobayashi, Yume; Tada, Shusuke; Seki, Masayuki; Enomoto, Takemi

    2014-09-12

    Highlights: • The UV sensitivity of POLH{sup −/−} cells was suppressed by disruption of WRNIP1. • In WRNIP1{sup −/−/−}/POLH{sup −/−} cells, mutation frequencies and SCE after irradiation reduced. • WRNIP1 defect recovered rate of fork progression after irradiation in POLH{sup −/−} cells. • WRNIP1 functions upstream of Polη in the translesion DNA synthesis pathway. - Abstract: WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH{sup −/−}) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  19. Cidofovir selectivity is based on the different response of normal and cancer cells to DNA damage

    PubMed Central

    2013-01-01

    Background Cidofovir (CDV) proved efficacious in treatment of human papillomaviruses (HPVs) hyperplasias. Antiproliferative effects of CDV have been associated with apoptosis induction, S-phase accumulation, and increased levels of tumor suppressor proteins. However, the molecular mechanisms for the selectivity and antitumor activity of CDV against HPV-transformed cells remain unexplained. Methods We evaluated CDV drug metabolism and incorporation into cellular DNA, in addition to whole genome gene expression profiling by means of microarrays in two HPV+ cervical carcinoma cells, HPV- immortalized keratinocytes, and normal keratinocytes. Results Determination of the metabolism and drug incorporation of CDV into genomic DNA demonstrated a higher rate of drug incorporation in HPV+ tumor cells and immortalized keratinocytes compared to normal keratinocytes. Gene expression profiling clearly showed distinct and specific drug effects in the cell types investigated. Although an effect on inflammatory response was seen in all cell types, different pathways were identified in normal keratinocytes compared to immortalized keratinocytes and HPV+ tumor cells. Notably, Rho GTPase pathways, LXR/RXR pathways, and acute phase response signaling were exclusively activated in immortalized cells. CDV exposed normal keratinocytes displayed activated cell cycle regulation upon DNA damage signaling to allow DNA repair via homologous recombination, resulting in genomic stability and survival. Although CDV induced cell cycle arrest in HPV- immortalized cells, DNA repair was not activated in these cells. In contrast, HPV+ cells lacked cell cycle regulation, leading to genomic instability and eventually apoptosis. Conclusions Taken together, our data provide novel insights into the mechanism of action of CDV and its selectivity for HPV-transformed cells. The proposed mechanism suggests that this selectivity is based on the inability of HPV+ cells to respond to DNA damage, rather than on a

  20. Interplay between Mdm2 and HIPK2 in the DNA damage response.

    PubMed

    Zhang, Xiao-Peng; Liu, Feng; Wang, Wei

    2014-07-01

    The tumour suppressor p53 is activated to induce cell-cycle arrest or apoptosis in the DNA damage response (DDR). p53 phosphorylation at Ser46 by HIPK2 (homeodomain-interacting protein kinase 2) is a critical event in apoptosis induction. Interestingly, HIPK2 is degraded by Mdm2 (a negative regulator of p53), whereas Mdm2 is downregulated by HIPK2 through several mechanisms. Here, we develop a four-module network model for the p53 pathway to clarify the role of interplay between Mdm2 and HIPK2 in the DDR evoked by ultraviolet radiation. By numerical simulations, we reveal that Mdm2-dependent HIPK2 degradation promotes cell survival after mild DNA damage and that inhibition of HIPK2 degradation is sufficient to trigger apoptosis. In response to severe damage, p53 phosphorylation at Ser46 is promoted by the accumulation of HIPK2 due to downregulation of nuclear Mdm2 in the later phase of the response. Meanwhile, the concentration of p53 switches from moderate to high levels, contributing to apoptosis induction. We show that the presence of three mechanisms for Mdm2 downregulation, i.e. repression of mdm2 expression, inhibition of its nuclear entry and HIPK2-induced degradation, guarantees the apoptosis of irreparably damaged cells. Our results agree well with multiple experimental observations, and testable predictions are also made. This work advances our understanding of the regulation of p53 activity in the DDR and suggests that HIPK2 should be a significant target for cancer therapy. PMID:24829283

  1. Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

    PubMed Central

    Sareen, Archana; Chaudhury, Indrajit; Adams, Nicole; Sobeck, Alexandra

    2012-01-01

    Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase. PMID:22753026

  2. Disruption of DNA Damage-Response by Propyl Gallate and 9-Aminoacridine.

    PubMed

    Matsuda, Shun; Matsuda, Yoko; Yanagisawa, Shin-Ya; Ikura, Masae; Ikura, Tsuyoshi; Matsuda, Tomonari

    2016-06-01

    The DNA-damage response (DDR) protects the genome from various types of endogenous and exogenous DNA damage, and can itself be a target of certain chemicals that give rise to chromosomal aberrations. Here, we developed a screening method to detect inhibition of Mediator of DNA damage Checkpoint 1 (MDC1) foci formation (the Enhanced Green Fluorescent Protein (EGFP)-MDC1 foci formation-inhibition assay) using EGFP-MDC1-expressing human cells. The assay identified propyl gallate (PG) and 9-aminoacridine (9-AA) as inhibitors of camptothecin (CPT)-induced MDC1 foci formation. We demonstrated that the inhibition of CPT-induced MDC1 foci formation by PG was caused by the direct suppression of histone H2AX phosphorylation at Ser139 (γH2AX), which is required for MDC1 foci formation, by quantifying γH2AX in cells and in vitro 9-AA also directly suppressed H2AX Ser139-phosphorylation in vitro but the concentration was much higher than that required to suppress CPT-induced MDC1 foci formation in cells. Consistent with these findings, PG and 9-AA both suppressed CPT-induced G2/M cell-cycle arrest and increased the number of abnormal nuclei. Our results suggest that early DDR-inhibitory effects of PG and 9-AA contribute to their chromosome-damaging potential, and that the EGFP-MDC1 foci formation-inhibition assay is useful for detection of and screening for H2AX Ser139-phosphorylation-inhibitory effects of chemicals. PMID:26928355

  3. Coordination of the Nuclear and Cytoplasmic Activities of p53 in Response to DNA Damage

    PubMed Central

    Pu, Tian; Zhang, Xiao-Peng; Liu, Feng; Wang, Wei

    2010-01-01

    The tumor suppressor p53 plays a key role in the cellular response to various stresses. Most previous studies have focused on either the nuclear or cytoplasmic proapoptotic functions of p53, ignoring the combination of both functions. To explore how the two functions of p53 are coordinated in the DNA damage response via computer simulation, we construct a model for the p53 network comprising coupled positive and negative feedback loops involving p53, Mdm2, and Akt, as well as PUMA and Bax. In our model p53 is stabilized and accumulates in the nucleus and cytoplasm upon DNA damage. Nuclear p53 induces expression of Mdm2, PTEN, PUMA, and Bax. Cytoplasmic p53 is then released from the p53·Bcl-xL complex by PUMA to activate Bax directly. We find that the switching between low and high protein levels underlies the decision between cell survival and death. Moreover, a balance between the nuclear and cytoplasmic p53 levels and appropriate levels of Akt and PUMA are required for reliable cell fate decision. Our results indicate that coordination of the transcription-dependent and -independent activities of p53 is important in determining cellular outcomes. These findings advance our understanding of the mechanism for p53-mediated cellular responses and provide clues to p53-based cancer therapy. PMID:20858413

  4. Protein kinase Cη activates NF-κB in response to camptothecin-induced DNA damage.

    PubMed

    Raveh-Amit, Hadas; Hai, Naama; Rotem-Dai, Noa; Shahaf, Galit; Gopas, Jacob; Livneh, Etta

    2011-08-26

    The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance. PMID:21820409

  5. Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells

    PubMed Central

    Nanya, Mai; Sato, Masaki; Tanimoto, Kousuke; Tozuka, Minoru; Mizutani, Shuki; Takagi, Masatoshi

    2015-01-01

    Etoposide, a topoisomerase 2 (TOP2) inhibitor, is associated with the development of KMT2A (MLL)-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL) rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL)-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP) assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq) identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll)-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective. PMID:26657054

  6. Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis.

    PubMed

    Jurado, Sabine; Smyth, Ian; van Denderen, Bryce; Tenis, Nora; Hammet, Andrew; Hewitt, Kimberly; Ng, Jane-Lee; McNees, Carolyn J; Kozlov, Sergei V; Oka, Hayato; Kobayashi, Masahiko; Conlan, Lindus A; Cole, Timothy J; Yamamoto, Ken-Ichi; Taniguchi, Yoshihito; Takeda, Shunichi; Lavin, Martin F; Heierhorst, Jörg

    2010-10-01

    Zn²(+)-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+)-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis. PMID:20975950

  7. Dual Functions of ASCIZ in the DNA Base Damage Response and Pulmonary Organogenesis

    PubMed Central

    Tenis, Nora; Hammet, Andrew; Hewitt, Kimberly; Ng, Jane-Lee; McNees, Carolyn J.; Kozlov, Sergei V.; Oka, Hayato; Kobayashi, Masahiko; Conlan, Lindus A.; Cole, Timothy J.; Yamamoto, Ken-ichi; Taniguchi, Yoshihito; Takeda, Shunichi; Lavin, Martin F.; Heierhorst, Jörg

    2010-01-01

    Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion or knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis. PMID:20975950

  8. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response.

    PubMed

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-01-01

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage. PMID:26238070

  9. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response

    PubMed Central

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-01-01

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage. PMID:26238070

  10. Characterization of LGALS3 (galectin-3) as a player in DNA damage response

    PubMed Central

    Carvalho, Renato S; Fernandes, Vanessa C; Nepomuceno, Thales C; Rodrigues, Deivid C; Woods, Nicholas T; Suarez-Kurtz, Guilherme; Chammas, Roger; Monteiro, Alvaro N; Carvalho, Marcelo A

    2014-01-01

    DNA damage repair (DDR) is an orchestrated process encompassing the injury detection to its complete resolution. DNA double-strand break lesions are repaired mainly by two distinct mechanisms: the error-free homologous recombination (HR) and the error-prone non-homologous end-joining. Galectin-3 (GAL3) is the unique member of the chimeric galectins subfamily and is reported to be involved in several cancer development and progression related events. Recently our group described a putative protein interaction between GAL3 and BARD1, the main partner of breast and ovarian cancer susceptibility gene product BRCA1, both involved in HR pathway. In this report we characterized GAL3/BARD1 protein interaction and evaluated the role of GAL3 in DDR pathways using GAL3 silenced human cells exposed to different DNA damage agents. In the absence of GAL3 we observed a delayed DDR response activation, as well as a decrease in the G2/M cell cycle checkpoint arrest associated with HR pathway. Moreover, using a TAP-MS approach we also determined the protein interaction network of GAL3. PMID:24755837

  11. A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus

    PubMed Central

    Shiraishi, Miyako; Ishino, Sonoko; Yamagami, Takeshi; Egashira, Yuriko; Kiyonari, Shinichi; Ishino, Yoshizumi

    2015-01-01

    DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3′ from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5′ from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea. PMID:25694513

  12. 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response.

    PubMed

    Martínez, Paula; Flores, Juana M; Blasco, Maria A

    2012-04-16

    TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)- and the ataxia telangiectasia and Rad3 related (ATR)-mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes. PMID:22508511

  13. Pim2 is important for regulating DNA damage response in multiple myeloma cells.

    PubMed

    Ramachandran, J; Santo, L; Siu, K T; Panaroni, C; Raje, N

    2016-01-01

    Pan proviral integrations of Moloney virus (PIM) inhibition in multiple myeloma (MM) results in reduced cell viability in tested human-derived MM cell lines and reduces tumor burden in xenograft mouse models, making PIMs important therapeutic targets for the disease. PIM kinase inhibitors are currently being tested clinically in MM. We sought to elucidate the role of the various PIMs in MM. Our data demonstrate that Pim2 has a significant role in MM cell cytotoxicity. Our data provide evidence for a novel role for Pim2 in the regulation of the DNA damage response (DDR). Knockdown of Pim2 upregulates several downstream DDR markers, mimicking the effects of doxorubicin (Dox) treatment of MM cells, and suggesting a role for the kinase as a negative regulator of this pathway. Dox-induced DNA damage results in a decrease in Pim2 levels, placing the kinase directly downstream of the site of Dox-DNA binding. Overexpression of Pim2 confers a slight survival advantage against Dox through antiapoptotic activity, further underscoring its relevance in the DDR pathway. These data provide insights into a novel mechanism of PIM kinase activity and provide the framework for designing therapeutic approaches in MM. PMID:27564460

  14. Forkhead transcription factor FoxF1 interacts with Fanconi anemia protein complexes to promote DNA damage response

    PubMed Central

    Pradhan, Arun; Ustiyan, Vladimir; Zhang, Yufang; Kalin, Tanya V.; Kalinichenko, Vladimir V.

    2016-01-01

    Forkhead box F1 (Foxf1) transcription factor is an important regulator of embryonic development but its role in tumor cells remains incompletely understood. While 16 proteins were characterized in Fanconi anemia (FA) core complex, its interactions with cellular transcriptional machinery remain poorly characterized. Here, we identified FoxF1 protein as a novel interacting partner of the FA complex proteins. Using multiple human and mouse tumor cell lines and Foxf1+/− mice we demonstrated that FoxF1 physically binds to and increases stability of FA proteins. FoxF1 co-localizes with FANCD2 in DNA repair foci in cultured cells and tumor tissues obtained from cisplatin-treated mice. In response to DNA damage, FoxF1-deficient tumor cells showed significantly reduced FANCD2 monoubiquitination and FANCM phosphorylation, resulting in impaired formation of DNA repair foci. FoxF1 knockdown caused chromosomal instability, nuclear abnormalities, and increased tumor cell death in response to DNA-damaging agents. Overexpression of FoxF1 in DNA-damaged cells improved stability of FA proteins, decreased chromosomal and nuclear aberrations, restored formation of DNA repair foci and prevented cell death after DNA damage. These findings demonstrate that FoxF1 is a key component of FA complexes and a critical mediator of DNA damage response in tumor cells. PMID:26625197

  15. Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells

    PubMed Central

    Puebla-Osorio, N; Damiani, E; Bover, L; Ullrich, S E

    2015-01-01

    Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2–M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment. PMID:25950475

  16. DNA Damage Response Is Involved in the Developmental Toxicity of Mebendazole in Zebrafish Retina.

    PubMed

    Sasagawa, Shota; Nishimura, Yuhei; Kon, Tetsuo; Yamanaka, Yukiko; Murakami, Soichiro; Ashikawa, Yoshifumi; Yuge, Mizuki; Okabe, Shiko; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Intestinal helminths cause iron-deficiency anemia in pregnant women, associated with premature delivery, low birth weight, maternal ill health, and maternal death. Although benzimidazole compounds such as mebendazole (MBZ) are highly efficacious against helminths, there are limited data on its use during pregnancy. In this study, we performed in vivo imaging of the retinas of zebrafish larvae exposed to MBZ, and found that exposure to MBZ during 2 and 3 days post-fertilization caused malformation of the retinal layers. To identify the molecular mechanism underlying the developmental toxicity of MBZ, we performed transcriptome analysis of zebrafish eyes. The analysis revealed that the DNA damage response was involved in the developmental toxicity of MBZ. We were also able to demonstrate that inhibition of ATM significantly attenuated the apoptosis induced by MBZ in the zebrafish retina. These results suggest that MBZ causes developmental toxicity in the zebrafish retina at least partly by activating the DNA damage response, including ATM signaling, providing a potential adverse outcome pathway in the developmental toxicity of MBZ in mammals. PMID:27014071

  17. DNA Damage Response Is Involved in the Developmental Toxicity of Mebendazole in Zebrafish Retina

    PubMed Central

    Sasagawa, Shota; Nishimura, Yuhei; Kon, Tetsuo; Yamanaka, Yukiko; Murakami, Soichiro; Ashikawa, Yoshifumi; Yuge, Mizuki; Okabe, Shiko; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Intestinal helminths cause iron-deficiency anemia in pregnant women, associated with premature delivery, low birth weight, maternal ill health, and maternal death. Although benzimidazole compounds such as mebendazole (MBZ) are highly efficacious against helminths, there are limited data on its use during pregnancy. In this study, we performed in vivo imaging of the retinas of zebrafish larvae exposed to MBZ, and found that exposure to MBZ during 2 and 3 days post-fertilization caused malformation of the retinal layers. To identify the molecular mechanism underlying the developmental toxicity of MBZ, we performed transcriptome analysis of zebrafish eyes. The analysis revealed that the DNA damage response was involved in the developmental toxicity of MBZ. We were also able to demonstrate that inhibition of ATM significantly attenuated the apoptosis induced by MBZ in the zebrafish retina. These results suggest that MBZ causes developmental toxicity in the zebrafish retina at least partly by activating the DNA damage response, including ATM signaling, providing a potential adverse outcome pathway in the developmental toxicity of MBZ in mammals. PMID:27014071

  18. Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response

    PubMed Central

    Bujdoso, Raymond; Landgraf, Matthias; Jackson, Walker S; Thackray, Alana M

    2015-01-01

    Protein misfolding neurodegenerative diseases arise through neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding-induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion-like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion-induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease. PMID:26279981

  19. miR-34 activity is modulated through 5'-end phosphorylation in response to DNA damage.

    PubMed

    Salzman, David W; Nakamura, Kotoka; Nallur, Sunitha; Dookwah, Michelle T; Metheetrairut, Chanatip; Slack, Frank J; Weidhaas, Joanne B

    2016-01-01

    MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5'-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5'-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level. PMID:26996824

  20. Renal-Retinal Ciliopathy Gene Sdccag8 Regulates DNA Damage Response Signaling

    PubMed Central

    Airik, Rannar; Slaats, Gisela G.; Guo, Zhi; Weiss, Anna-Carina; Khan, Naheed; Ghosh, Amiya; Hurd, Toby W.; Bekker-Jensen, Simon; Schrøder, Jacob M.; Elledge, Steve J.; Andersen, Jens S.; Kispert, Andreas; Castelli, Maddalena; Boletta, Alessandra; Giles, Rachel H.

    2014-01-01

    Nephronophthisis-related ciliopathies (NPHP-RCs) are developmental and degenerative kidney diseases that are frequently associated with extrarenal pathologies such as retinal degeneration, obesity, and intellectual disability. We recently identified mutations in a gene encoding the centrosomal protein SDCCAG8 as causing NPHP type 10 in humans. To study the role of Sdccag8 in disease pathogenesis, we generated a Sdccag8 gene-trap mouse line. Homozygous Sdccag8gt/gt mice lacked the wild-type Sdccag8 transcript and protein, and recapitulated the human phenotypes of NPHP and retinal degeneration. These mice exhibited early onset retinal degeneration that was associated with rhodopsin mislocalization in the photoreceptors and reduced cone cell numbers, and led to progressive loss of vision. By contrast, renal histologic changes occurred later, and no global ciliary defects were observed in the kidneys. Instead, renal pathology was associated with elevated levels of DNA damage response signaling activity. Cell culture studies confirmed the aberrant activation of DNA damage response in Sdccag8gt/gt-derived cells, characterized by elevated levels of γH2AX and phosphorylated ATM and cell cycle profile abnormalities. Our analysis of Sdccag8gt/gt mice indicates that the pleiotropic phenotypes in these mice may arise through multiple tissue-specific disease mechanisms. PMID:24722439

  1. LEM-3 – A LEM Domain Containing Nuclease Involved in the DNA Damage Response in C. elegans

    PubMed Central

    Dittrich, Christina M.; Kratz, Katja; Sendoel, Ataman; Gruenbaum, Yosef; Jiricny, Josef; Hengartner, Michael O.

    2012-01-01

    The small nematode Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage – possibly by promoting the reorganization of damaged chromatin. PMID:22383942

  2. Mechanisms of Hg species induced toxicity in cultured human astrocytes: genotoxicity and DNA-damage response.

    PubMed

    Pieper, Imke; Wehe, Christoph A; Bornhorst, Julia; Ebert, Franziska; Leffers, Larissa; Holtkamp, Michael; Höseler, Pia; Weber, Till; Mangerich, Aswin; Bürkle, Alexander; Karst, Uwe; Schwerdtle, Tanja

    2014-03-01

    The toxicologically most relevant mercury (Hg) species for human exposure is methylmercury (MeHg). Thiomersal is a common preservative used in some vaccine formulations. The aim of this study is to get further mechanistic insight into the yet not fully understood neurotoxic modes of action of organic Hg species. Mercury species investigated include MeHgCl and thiomersal. Additionally HgCl2 was studied, since in the brain mercuric Hg can be formed by dealkylation of the organic species. As a cellular system astrocytes were used. In vivo astrocytes provide the environment necessary for neuronal function. In the present study, cytotoxic effects of the respective mercuricals increased with rising alkylation level and correlated with their cellular bioavailability. Further experiments revealed for all species at subcytotoxic concentrations no induction of DNA strand breaks, whereas all species massively increased H2O2-induced DNA strand breaks. This co-genotoxic effect is likely due to a disturbance of the cellular DNA damage response. Thus, at nanomolar, sub-cytotoxic concentrations, all three mercury species strongly disturbed poly(ADP-ribosyl)ation, a signalling reaction induced by DNA strand breaks. Interestingly, the molecular mechanism behind this inhibition seems to be different for the species. Since chronic PARP-1 inhibition is also discussed to sacrifice neurogenesis and learning abilities, further experiments on neurons and in vivo studies could be helpful to clarify whether the inhibition of poly(ADP-ribosyl)ation contributes to organic Hg induced neurotoxicity. PMID:24549367

  3. DNA damage checkpoints in mammals.

    PubMed

    Niida, Hiroyuki; Nakanishi, Makoto

    2006-01-01

    DNA damage is a common event and probably leads to mutation or deletion within chromosomal DNA, which may cause cancer or premature aging. DNA damage induces several cellular responses including DNA repair, checkpoint activity and the triggering of apoptotic pathways. DNA damage checkpoints are associated with biochemical pathways that end delay or arrest of cell-cycle progression. These checkpoints engage damage sensor proteins, such as the Rad9-Rad1-Hus1 (9-1-1) complex, and the Rad17-RFC complex, in the detection of DNA damage and transduction of signals to ATM, ATR, Chk1 and Chk2 kinases. Chk1 and Chk2 kinases regulate Cdc25, Wee1 and p53 that ultimately inactivate cyclin-dependent kinases (Cdks) which inhibit cell-cycle progression. In this review, we discuss the molecular mechanisms by which DNA damage is recognized by sensor proteins and signals are transmitted to Cdks. We classify the genes involved in checkpoint signaling into four categories, namely sensors, mediators, transducers and effectors, although their proteins have the broad activity, and thus this classification is for convenience and is not definitive. PMID:16314342

  4. Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

    PubMed Central

    Shin, David S.; Pratt, Ashley J.; Tainer, John A.

    2014-01-01

    As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine. PMID:24701133

  5. Maximiscin Induces DNA Damage, Activates DNA Damage Response Pathways, and Has Selective Cytotoxic Activity against a Subtype of Triple-Negative Breast Cancer.

    PubMed

    Robles, Andrew J; Du, Lin; Cichewicz, Robert H; Mooberry, Susan L

    2016-07-22

    Triple-negative breast cancers are highly aggressive, and patients with these types of tumors have poor long-term survival. These breast cancers do not express estrogen or progesterone receptors and do not have gene amplification of human epidermal growth factor receptor 2; therefore, they do not respond to available targeted therapies. The lack of targeted therapies for triple-negative breast cancers stems from their heterogeneous nature and lack of a clear definition of driver defects. Studies have recently identified triple-negative breast cancer molecular subtypes based on gene expression profiling and representative cell lines, allowing for the identification of subtype-specific drug leads and molecular targets. We previously reported the identification of a new fungal metabolite named maximiscin (1) identified through a crowdsourcing program. New results show that 1 has selective cytotoxic efficacy against basal-like 1 MDA-MB-468 cells compared to cell lines modeling other triple-negative breast cancer molecular subtypes. This compound also exhibited antitumor efficacy in a xenograft mouse model. The mechanisms of action of 1 in MDA-MB-468 cells were investigated to identify potential molecular targets and affected pathways. Compound 1 caused accumulation of cells in the G1 phase of the cell cycle, suggesting induction of DNA damage. Indeed, treatment with 1 caused DNA double-strand breaks with concomitant activation of the DNA damage response pathways, indicated by phosphorylation of p53, Chk1, and Chk2. Collectively, these results suggest basal-like triple-negative breast cancer may be inherently sensitive to DNA-damaging agents relative to other triple-negative breast cancer subtypes. These results also demonstrate the potential of our citizen crowdsourcing program to identify new lead molecules for treating the subtypes of triple-negative breast cancer. PMID:27310425

  6. The p53 network: Cellular and systemic DNA damage responses in aging and cancer

    PubMed Central

    Reinhardt, H. Christian; Schumacher, Björn

    2014-01-01

    Genome instability contributes to cancer development and accelerates age-related pathologies as evidenced by a variety of congenital cancer susceptibility and progeroid syndromes that are caused by defects in genome maintenance mechanisms. DNA damage response pathways that are mediated through the tumor suppressor p53 play an important role in the cell intrinsic responses to genome instability, including a transient cell cycle arrest, senescence and apoptosis. Both senescence and apoptosis are powerful tumor suppressive pathways preventing the uncontrolled proliferation of transformed cells. However, both pathways can potentially deplete stem and progenitor cell pools, thus promoting tissue degeneration and organ failure, which are both hallmarks of aging. p53 signaling is also involved in mediating non-cell autonomous interactions with the innate immune system and in the systemic adjustments during the aging process. The network of p53 target genes thus functions as an important regulator of cancer prevention and the physiology of aging. PMID:22265392

  7. Growth factor independence-1 antagonizes a p53-induced DNA damage response pathway in lymphoblastic leukemia

    PubMed Central

    Khandanpour, Cyrus; Phelan, James D.; Vassen, Lothar; Schütte, Judith; Chen, Riyan; Horman, Shane R.; Gaudreau, Marie-Claude; Krongold, Joseph; Zhu, Jinfang; Paul, William E.; Dührsen, Ulrich; Göttgens, Bertie; Grimes, H. Leighton; Möröy, Tarik

    2013-01-01

    Summary Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Since oncogenic signaling activates a p53-dependent DNA-damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function, since Gfi1 ablation exacerbates p53 responses, and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of pro-apoptotic p53 targets such as Bax, Noxa (Pmaip1) and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias. PMID:23410974

  8. Targeting DNA Damage Response in the Radio(Chemo)therapy of Non-Small Cell Lung Cancer

    PubMed Central

    Li, Ling; Zhu, Tao; Gao, Yuan-Feng; Zheng, Wei; Wang, Chen-Jing; Xiao, Ling; Huang, Ma-Sha; Yin, Ji-Ye; Zhou, Hong-Hao; Liu, Zhao-Qian

    2016-01-01

    Lung cancer is the leading cause of cancer death worldwide due to its high incidence and mortality. As the most common lung cancer, non-small cell lung cancer (NSCLC) is a terrible threat to human health. Despite improvements in diagnosis and combined treatments including surgical resection, radiotherapy and chemotherapy, the overall survival for NSCLC patients still remains poor. DNA damage is considered to be the primary cause of lung cancer development and is normally recognized and repaired by the intrinsic DNA damage response machinery. The role of DNA repair pathways in radio(chemo)therapy-resistant cancers has become an area of significant interest in the clinical setting. Meanwhile, some studies have proved that genetic and epigenetic factors can alter the DNA damage response and repair, which results in changes of the radiation and chemotherapy curative effect in NSCLC. In this review, we focus on the effect of genetic polymorphisms and epigenetic factors such as miRNA regulation and lncRNA regulation participating in DNA damage repair in response to radio(chemo)therapy in NSCLC. These may provide novel information on the radio(chemo)therapy of NSCLC based on the individual DNA damage response. PMID:27258253

  9. Ubiquitin-SUMO Circuitry Controls Activated Fanconi Anemia ID Complex Dosage in Response to DNA Damage

    PubMed Central

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T.; Passmore, Lori A.; Patel, Ketan J.; Olsen, Jesper V.; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    Summary We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage. PMID:25557546

  10. Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation

    PubMed Central

    Ogrunc, M; Di Micco, R; Liontos, M; Bombardelli, L; Mione, M; Fumagalli, M; Gorgoulis, V G; d'Adda di Fagagna, F

    2014-01-01

    Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents. PMID:24583638