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Sample records for damage response dna

  1. Autophagy in DNA damage response.

    PubMed

    Czarny, Piotr; Pawlowska, Elzbieta; Bialkowska-Warzecha, Jolanta; Kaarniranta, Kai; Blasiak, Janusz

    2015-01-23

    DNA damage response (DDR) involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1). mTORC1 represses autophagy via phosphorylation of the ULK1/2-Atg13-FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADP)ribose polymerase 1 (PARP-1), Mre11-Rad50-Nbs1 (MRN) complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  2. Sphingolipids in the DNA Damage Response

    PubMed Central

    Carroll, Brittany; Donaldson, Cat; Obeid, Lina

    2014-01-01

    Recently, sphingolipid metabolizing enzymes have emerged as important targets of many chemotherapeutics and DNA damaging agents and therefore play significant roles in mediating the physiological response of the cell to DNA damage. In this review we will highlight points of connection between the DNA damage response (DDR) and sphingolipid metabolism; specifically how certain sphingolipid enzymes are regulated in response to DNA damage and how the bioactive lipids produced by these enzymes affect cell fate. PMID:25434743

  3. 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.

  4. DNA damage response, redox status and hematopoiesis.

    PubMed

    Weiss, Cary N; Ito, Keisuke

    2014-01-01

    The ability of hematopoietic stem cells (HSCs) to self-renew and differentiate into progenitors is essential for homeostasis of the hematopoietic system. The longevity of HSCs makes them vulnerable to accumulating DNA damage, which may be leukemogenic or result in senescence and cell death. Additionally, the ability of HSCs to self-renew and differentiate allows DNA damage to spread throughout the hematologic system, leaving the organism vulnerable to disease. In this review we discuss cell fate decisions made in the face of DNA damage and other cellular stresses, and the role of reactive oxygen species in the long-term maintenance of HSCs and their DNA damage response.

  5. The DNA damage response during mitosis.

    PubMed

    Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

    2013-10-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

  6. 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

  7. 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.

  8. Historical Perspective on the DNA Damage Response

    PubMed Central

    Hanawalt, Philip C.

    2015-01-01

    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

  9. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

    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.

  10. Targeting the DNA Damage Response in Cancer.

    PubMed

    O'Connor, Mark J

    2015-11-19

    An underlying hallmark of cancers is their genomic instability, which is associated with a greater propensity to accumulate DNA damage. Historical treatment of cancer by radiotherapy and DNA-damaging chemotherapy is based on this principle, yet it is accompanied by significant collateral damage to normal tissue and unwanted side effects. Targeted therapy based on inhibiting the DNA damage response (DDR) in cancers offers the potential for a greater therapeutic window by tailoring treatment to patients with tumors lacking specific DDR functions. The recent approval of olaparib (Lynparza), the poly (ADP-ribose) polymerase (PARP) inhibitor for treating tumors harboring BRCA1 or BRCA2 mutations, represents the first medicine based on this principle, exploiting an underlying cause of tumor formation that also represents an Achilles' heel. This review highlights the different concepts behind targeting DDR in cancer and how this can provide significant opportunities for DDR-based therapies in the future.

  11. 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.

  12. Immunofluorescence Imaging of DNA Damage Response Proteins

    PubMed Central

    Bennett, Brian T.; Bewersdorf, Jörg; Knight, Kendall L.

    2013-01-01

    Immunofluorescence imaging has provided captivating visual evidence for numerous cellular events, from vesicular trafficking, organelle maturation and cell division to nuclear processes including the appearance of various proteins and chromatin components in distinct foci in response to DNA damaging agents. With the advent of new super-resolution microscope technologies such as 4Pi microscopy, standard immunofluorescence protocols deserve some reevaluation in order to take full advantage of these new technological accomplishments. Here we describe several methodological considerations that will help overcome some of the limitations that may result from the use of currently applied procedures, with particular attention paid to the analysis of possible colocalization of fluorescent signals. We conclude with an example of how application of optimized methods led to a breakthrough in super-resolution imaging of nuclear events occurring in response to DNA damage. PMID:19245833

  13. 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

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

    PubMed

    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.

  15. DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs.

    PubMed

    Rossiello, Francesca; Aguado, Julio; Sepe, Sara; Iannelli, Fabio; Nguyen, Quan; Pitchiaya, Sethuramasundaram; Carninci, Piero; d'Adda di Fagagna, Fabrizio

    2017-02-27

    The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.

  16. Telomeres, histone code, and DNA damage response.

    PubMed

    Misri, S; Pandita, S; Kumar, R; Pandita, T K

    2008-01-01

    Genomic stability is maintained by telomeres, the end terminal structures that protect chromosomes from fusion or degradation. Shortening or loss of telomeric repeats or altered telomere chromatin structure is correlated with telomere dysfunction such as chromosome end-to-end associations that could lead to genomic instability and gene amplification. The structure at the end of telomeres is such that its DNA differs from DNA double strand breaks (DSBs) to avoid nonhomologous end-joining (NHEJ), which is accomplished by forming a unique higher order nucleoprotein structure. Telomeres are attached to the nuclear matrix and have a unique chromatin structure. Whether this special structure is maintained by specific chromatin changes is yet to be thoroughly investigated. Chromatin modifications implicated in transcriptional regulation are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. The histone code in which distinct histone tail-protein interactions promote engagement may be the deciding factor for choosing specific DSB repair pathways. Recent evidence suggests that such mechanisms are involved in DNA damage detection and repair. Altered telomere chromatin structure has been linked to defective DNA damage response (DDR), and eukaryotic cells have evolved DDR mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. Recent studies suggest that chromatin modifying factors play a critical role in the maintenance of genomic stability. This review will summarize the role of DNA damage repair proteins specifically ataxia-telangiectasia mutated (ATM) and its effectors and the telomere complex in maintaining genome stability.

  17. DNA mismatch repair and the DNA damage response

    PubMed Central

    Li, Zhongdao; Pearlman, Alexander H.; Hsieh, Peggy

    2015-01-01

    This review discusses the role of DNA mismatch repair (MMR) in the DNA damage response (DDR) that triggers cell cycle arrest and, in some cases, apoptosis. Although the focus is on findings from mammalian cells, much has been learned from studies in other organisms including bacteria and yeast [1,2]. MMR promotes a DDR mediated by a key signaling kinase, ATM and Rad3-related (ATR), in response to various types of DNA damage including some encountered in widely used chemotherapy regimes. An introduction to the DDR mediated by ATR reveals its immense complexity and highlights the many biological and mechanistic questions that remain. Recent findings and future directions are highlighted. PMID:26704428

  18. Profiling DNA damage response following mitotic perturbations

    PubMed Central

    S. Pedersen, Ronni; Karemore, Gopal; Gudjonsson, Thorkell; Rask, Maj-Britt; Neumann, Beate; Hériché, Jean-Karim; Pepperkok, Rainer; Ellenberg, Jan; Gerlich, Daniel W.; Lukas, Jiri; Lukas, Claudia

    2016-01-01

    Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes. PMID:27976684

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

    PubMed Central

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

    2015-01-01

    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

  20. 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

  1. Replication fork dynamics and the DNA damage response.

    PubMed

    Jones, Rebecca M; Petermann, Eva

    2012-04-01

    Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

  2. Systemic DNA damage responses in aging and diseases

    PubMed Central

    Ribezzo, Flavia; Shiloh, Yosef; Schumacher, Björn

    2016-01-01

    The genome is constantly attacked by a variety of genotoxic insults. The causal role for DNA damage in aging and cancer is exemplified by genetic defects in DNA repair that underlie a broad spectrum of acute and chronic human disorders that are characterized by developmental abnormalities, premature aging, and cancer predisposition. The disease symptoms are typically tissue-specific with uncertain genotype-phenotype correlation. The cellular DNA damage response (DDR) has been extensively investigated ever since yeast geneticists discovered DNA damage checkpoint mechanisms, several decades ago. In recent years, it has become apparent that not only cell-autonomous but also systemic DNA damage responses determine the outcome of genome instability in organisms. Understanding the mechanisms of non-cell-autonomous DNA damage responses will provide important new insights into the role of genome instability in human aging and a host of diseases including cancer and might better explain the complex phenotypes caused by genome instability. PMID:26773346

  3. 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.

  4. CHK2 kinase in the DNA damage response and beyond

    PubMed Central

    Zannini, Laura; Delia, Domenico; Buscemi, Giacomo

    2014-01-01

    The serine/threonine kinase CHK2 is a key component of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates >20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In particular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy. PMID:25404613

  5. DNA Damage Response and Autophagy: A Meaningful Partnership

    PubMed Central

    Eliopoulos, Aristides G.; Havaki, Sophia; Gorgoulis, Vassilis G.

    2016-01-01

    Autophagy and the DNA damage response (DDR) are biological processes essential for cellular and organismal homeostasis. Herein, we summarize and discuss emerging evidence linking DDR to autophagy. We highlight published data suggesting that autophagy is activated by DNA damage and is required for several functional outcomes of DDR signaling, including repair of DNA lesions, senescence, cell death, and cytokine secretion. Uncovering the mechanisms by which autophagy and DDR are intertwined provides novel insight into the pathobiology of conditions associated with accumulation of DNA damage, including cancer and aging, and novel concepts for the development of improved therapeutic strategies against these pathologies. PMID:27917193

  6. 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.

  7. DNA damage response to the Mdm2 inhibitor Nutlin-3

    PubMed Central

    Verma, Rajeev; Rigatti, Marc J.; Belinsky, Glenn S.; Godman, Cassandra A.; Giardina, Charles

    2009-01-01

    Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethane-induced mouse AJ02-NM0 colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin). Quantification of γH2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/M were mounting a DNA damage response (compared to a ~50% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events. PMID:19788889

  8. 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.

  9. 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

  10. Activation of DNA damage response signaling by condensed chromatin.

    PubMed

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

  11. 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

  12. 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.

  13. Multiomic Analysis of the UV-Induced DNA Damage Response.

    PubMed

    Boeing, Stefan; Williamson, Laura; Encheva, Vesela; Gori, Ilaria; Saunders, Rebecca E; Instrell, Rachael; Aygün, Ozan; Rodriguez-Martinez, Marta; Weems, Juston C; Kelly, Gavin P; Conaway, Joan W; Conaway, Ronald C; Stewart, Aengus; Howell, Michael; Snijders, Ambrosius P; Svejstrup, Jesper Q

    2016-05-11

    In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response.

  14. 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

  15. 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

  16. DNA damage response during mouse oocyte maturation

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  17. Detecting ATM-Dependent Chromatin Modification in DNA Damage Response

    PubMed Central

    Udayakumar, Durga; Horikoshi, Nobuo; Mishra, Lope; Hunt, Clayton; Pandita, Tej K.

    2015-01-01

    Loss of function or mutation of the ataxia–telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage. PMID:25827888

  18. DNA damage response defect in Williams-Beuren syndrome

    PubMed Central

    Guenat, David; Merla, Giuseppe; Deconinck, Eric; Borg, Christophe; Rohrlich, Pierre-Simon

    2017-01-01

    Williams-Beuren syndrome (WBS, no. OMIM 194050) is a rare multisystem genetic disorder caused by a microdeletion on chromosome 7q11.23 and characterized by cardiovascular malformations, mental retardation, and a specific facial dysmorphism. Recently, we reported that a series of non-Hodgkin's lymphoma occurs in children with WBS and thus hypothesized that a predisposition to cancer may be associated with this genetic disorder. The aim of the present study was to ascertain the role played by three genes hemizygously deleted in WBS (RFC2, GTF2I and BAZ1B) in DNA damage response pathways. Cell proliferation, cell cycle analysis, γ-H2A.X induction, and expression of DNA damage response proteins were investigated upon exposure to genotoxic treatments in WBS patient-derived primary fibroblasts and in the 293T cell line treated with specific siRNAs targeting RFC2, GTF2I and BAZ1B. An impaired hydroxyurea-induced phosphorylation of CHK1 was observed in the WBS cells. However, this defective DNA damage response was not associated with an increased sensitivity to genotoxic agents. In addition, depletion of RFC2, GTF2I and BAZ1B using specific siRNAs did not have a significant impact on the DNA damage response in 293T cells. Our results highlight that the ATR-dependent DNA damage response is impaired in WBS patient cells but is also dispensable for viability when these cells undergo a genotoxic stress. The mechanism by which the ATR pathway is impaired in WBS warrants elucidation through further investigation. PMID:28098859

  19. Viruses and the DNA Damage Response: Activation and Antagonism.

    PubMed

    Luftig, Micah A

    2014-11-01

    Viruses must interact with their hosts in order to replicate; these interactions often provoke the evolutionarily conserved response to DNA damage, known as the DNA damage response (DDR). The DDR can be activated by incoming viral DNA, during the integration of retroviruses, or in response to the aberrant DNA structures generated upon replication of DNA viruses. Furthermore, DNA and RNA viral proteins can induce the DDR by promoting inappropriate S phase entry, by modifying cellular DDR factors directly, or by unintentionally targeting host DNA. The DDR may be antiviral, although viruses often require proximal DDR activation of repair and recombination factors to facilitate replication as well as downstream DDR signaling suppression to ensure cell survival. An unintended consequence of DDR attenuation during infection is the long-term survival and proliferation of precancerous cells. Therefore, the molecular basis for DDR activation and attenuation by viruses remains an important area of study that will likely provide key insights into how viruses have evolved with their hosts.

  20. DNA damage response in nephrotoxic and ischemic kidney injury.

    PubMed

    Yan, Mingjuan; Tang, Chengyuan; Ma, Zhengwei; Huang, Shuang; Dong, Zheng

    2016-10-27

    DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore relies on a thorough elucidation of the DDR pathways in various forms of AKI.

  1. MicroRNAs, DNA Damage Response, and Cancer Treatment

    PubMed Central

    He, Mingyang; Zhou, Weiwei; Li, Chuang; Guo, Mingxiong

    2016-01-01

    As a result of various stresses, lesions caused by DNA-damaging agents occur constantly in each cell of the human body. Generally, DNA damage is recognized and repaired by the DNA damage response (DDR) machinery, and the cells survive. When repair fails, the genomic integrity of the cell is disrupted—a hallmark of cancer. In addition, the DDR plays a dual role in cancer development and therapy. Cancer radiotherapy and chemotherapy are designed to eliminate cancer cells by inducing DNA damage, which in turn can promote tumorigenesis. Over the past two decades, an increasing number of microRNAs (miRNAs), small noncoding RNAs, have been identified as participating in the processes regulating tumorigenesis and responses to cancer treatment with radiation therapy or genotoxic chemotherapies, by modulating the DDR. The purpose of this review is to summarize the recent findings on how miRNAs regulate the DDR and discuss the therapeutic functions of miRNAs in cancer in the context of DDR regulation. PMID:27973455

  2. 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

  3. 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

  4. Targeting ATR in DNA damage response and cancer therapeutics.

    PubMed

    Fokas, Emmanouil; Prevo, Remko; Hammond, Ester M; Brunner, Thomas B; McKenna, W Gillies; Muschel, Ruth J

    2014-02-01

    The ataxia telangiectasia and Rad3-related (ATR) plays an important role in maintaining genome integrity during DNA replication through the phosphorylation and activation of Chk1 and regulation of the DNA damage response. Preclinical studies have shown that disruption of ATR pathway can exacerbate the levels of replication stress in oncogene-driven murine tumors to promote cell killing. Additionally, inhibition of ATR can sensitise tumor cells to radiation or chemotherapy. Accumulating evidence suggests that targeting ATR can selectively sensitize cancer cells but not normal cells to DNA damage. Furthermore, in hypoxic conditions, ATR blockade results in overloading replication stress and DNA damage response causing cell death. Despite the attractiveness of ATR inhibition in the treatment of cancer, specific ATR inhibitors have remained elusive. In the last two years however, selective ATR inhibitors suitable for in vitro and - most recently - in vivo studies have been identified. In this article, we will review the literature on ATR function, its role in DDR and the potential of ATR inhibition to enhance the efficacy of radiation and chemotherapy.

  5. Rewiring of genetic networks in response to DNA damage.

    PubMed

    Bandyopadhyay, Sourav; Mehta, Monika; Kuo, Dwight; Sung, Min-Kyung; Chuang, Ryan; Jaehnig, Eric J; Bodenmiller, Bernd; Licon, Katherine; Copeland, Wilbert; Shales, Michael; Fiedler, Dorothea; Dutkowski, Janusz; Guénolé, Aude; van Attikum, Haico; Shokat, Kevan M; Kolodner, Richard D; Huh, Won-Ki; Aebersold, Ruedi; Keogh, Michael-Christopher; Krogan, Nevan J; Ideker, Trey

    2010-12-03

    Although cellular behaviors are dynamic, the networks that govern these behaviors have been mapped primarily as static snapshots. Using an approach called differential epistasis mapping, we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases, and transcription factors as the cell responds to DNA damage. Differential interactions uncover many gene functions that go undetected in static conditions. They are very effective at identifying DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally stable in response to perturbation, but the functional relations between these complexes are substantially reorganized. Differential networks chart a new type of genetic landscape that is invaluable for mapping cellular responses to stimuli.

  6. The Intra-S Checkpoint Responses to DNA Damage

    PubMed Central

    Iyer, Divya Ramalingam; Rhind, Nicholas

    2017-01-01

    Faithful duplication of the genome is a challenge because DNA is susceptible to damage by a number of intrinsic and extrinsic genotoxins, such as free radicals and UV light. Cells activate the intra-S checkpoint in response to damage during S phase to protect genomic integrity and ensure replication fidelity. The checkpoint prevents genomic instability mainly by regulating origin firing, fork progression, and transcription of G1/S genes in response to DNA damage. Several studies hint that regulation of forks is perhaps the most critical function of the intra-S checkpoint. However, the exact role of the checkpoint at replication forks has remained elusive and controversial. Is the checkpoint required for fork stability, or fork restart, or to prevent fork reversal or fork collapse, or activate repair at replication forks? What are the factors that the checkpoint targets at stalled replication forks? In this review, we will discuss the various pathways activated by the intra-S checkpoint in response to damage to prevent genomic instability. PMID:28218681

  7. Breaking the DNA damage response to improve cervical cancer treatment.

    PubMed

    Wieringa, Hylke W; van der Zee, Ate G J; de Vries, Elisabeth G E; van Vugt, Marcel A T M

    2016-01-01

    Every year, cervical cancer affects ∼500,000 women worldwide, and ∼275,000 patients die of this disease. The addition of platin-based chemotherapy to primary radiotherapy has increased 5-year survival of advanced-stage cervical cancer patients, which is, however, still only 66%. One of the factors thought to contribute to treatment failure is the ability of tumor cells to repair chemoradiotherapy-induced DNA damage. Therefore, sensitization of tumor cells for chemoradiotherapy via inhibition of the DNA damage response (DDR) as a novel strategy to improve therapy effect, is currently studied pre-clinically as well as in the clinic. Almost invariably, cervical carcinogenesis involves infection with the human papillomavirus (HPV), which inactivates part of the DNA damage response. This HPV-mediated partial inactivation of the DDR presents therapeutic targeting of the residual DDR as an interesting approach to achieve chemoradio-sensitization for cervical cancer. How the DDR can be most efficiently targeted, however, remains unclear. The fact that cisplatin and radiotherapy activate multiple signaling axes within the DDR further complicates a rational choice of therapeutic targets within the DDR. In this review, we provide an overview of the current preclinical and clinical knowledge about targeting the DDR in cervical cancer.

  8. 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

  9. 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

  10. HSV-I and the cellular DNA damage response.

    PubMed

    Smith, Samantha; Weller, Sandra K

    2015-04-01

    Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al. that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

  11. ATM kinase: Much more than a DNA damage responsive protein.

    PubMed

    Guleria, Ayushi; Chandna, Sudhir

    2016-03-01

    ATM, mutation of which causes Ataxia telangiectasia, has emerged as a cardinal multifunctional protein kinase during past two decades as evidenced by various studies from around the globe. Further to its well established and predominant role in DNA damage response, ATM has also been understood to help in maintaining overall functional integrity of cells; since its mutation, inactivation or deficiency results in a variety of pathological manifestations besides DNA damage. These include oxidative stress, metabolic syndrome, mitochondrial dysfunction as well as neurodegeneration. Recently, high throughput screening using proteomics, metabolomics and transcriptomic studies revealed several proteins which might be acting as substrates of ATM. Studies that can help in identifying effective regulatory controls within the ATM-mediated pathways/mechanisms can help in developing better therapeutics. In fact, more in-depth understanding of ATM-dependent cellular signals could also help in the treatment of variety of other disease conditions since these pathways seem to control many critical cellular functions. In this review, we have attempted to put together a detailed yet lucid picture of the present-day understanding of ATM's role in various pathophysiological conditions involving DNA damage and beyond.

  12. Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response.

    PubMed

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

    2009-07-21

    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.

  13. DNA damage response and sphingolipid signaling in liver diseases

    PubMed Central

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

    2016-01-01

    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. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

    PubMed Central

    Nelson, W G; Kastan, M B

    1994-01-01

    The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging

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

    PubMed Central

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

    2016-01-01

    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

  16. Circadian Clock Control of the Cellular Response to DNA Damage

    PubMed Central

    Sancar, Aziz; Lindsey-Boltz, Laura A.; Kang, Tae-Hong; Reardon, Joyce T.; Lee, Jin Hyup; Ozturk, Nuri

    2010-01-01

    Mammalian cells possess a cell-autonomous molecular clock which controls the timing of many biochemical reactions and hence the cellular response to environmental stimuli including genotoxic stress. The clock consists of an autoregulatory transcription-translation feedback loop made up of four genes/proteins, BMal1, Clock, Cryptochrome, and Period. The circadian clock has an intrinsic period of about 24 hours, and it dictates the rates of many biochemical reactions as a function of the time of the day. Recently, it has become apparent that the circadian clock plays an important role in determining the strengths of cellular responses to DNA damage including repair, checkpoints, and apoptosis. These new insights are expected to guide development of novel mechanism-based chemotherapeutic regimens. PMID:20227409

  17. Host DNA damage response facilitates African swine fever virus infection.

    PubMed

    Simões, Margarida; Martins, Carlos; Ferreira, Fernando

    2013-07-26

    Studies with different viral infection models on virus interactions with the host cell nucleus have opened new perspectives on our understanding of the molecular basis of these interactions in African swine fever virus (ASFV) infection. The present study aims to characterize the host DNA damage response (DDR) occurring upon in vitro infection with the ASFV-Ba71V isolate. We evaluated protein levels during ASFV time-course infection, of several signalling cascade factors belonging to DDR pathways involved in double strand break repair - Ataxia Telangiectasia Mutated (ATM), ATM-Rad 3 related (ATR) and DNA dependent protein kinase catalytic subunit (DNA-PKcs). DDR inhibitory trials using caffeine and wortmannin and ATR inducible-expression cell lines were used to confirm specific pathway activation during viral infection. Our results show that ASFV specifically elicits ATR-mediated pathway activation from the early phase of infection with increased levels of H2AX, RPA32, p53, ATR and Chk1 phosphorylated forms. Viral p72 synthesis was abrogated by ATR kinase inhibitors and also in ATR-kd cells. Furthermore, a reduction of viral progeny was identified in these cells when compared to the outcome of infection in ATR-wt. Overall, our results strongly suggest that the ATR pathway plays an essential role for successful ASFV infection of host cells.

  18. 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

  19. RAD54 forms DNA repair foci in response to DNA damage in living plant cells.

    PubMed

    Hirakawa, Takeshi; Hasegawa, Junko; White, Charles I; Matsunaga, Sachihiro

    2017-02-02

    Plants have various defense mechanisms against environmental stresses that induce DNA damage. Genetic and biochemical analyses have revealed the sensing and signaling of DNA damage, but little is known about subnuclear dynamics in response to DNA damage in living plant cells. Here, we observed that the chromatin remodeling factor RAD54, which is involved in DNA repair via the homologous recombination pathway, formed subnuclear foci (termed RAD54 foci) in Arabidopsis thaliana after induction of DNA double-strand breaks. The appearance of RAD54 foci was dependent on the ATAXIA-TELANGIECTASIA MUTATED-SUPPRESSOR OF GAMMA RESPONSE 1 pathway, and RAD54 foci were co-localized with γH2AX signals. Laser irradiation of a subnuclear area demonstrated that in living cells RAD54 was specifically accumulated at the damaged site. In addition, the formation of RAD54 foci showed specificity for cell type and region. We conclude that RAD54 foci correspond to DNA repair foci in A. thaliana.

  20. 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

  1. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

    PubMed Central

    Polo, Sophie E.; Jackson, Stephen P.

    2011-01-01

    Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR. PMID:21363960

  2. Retinoblastoma loss modulates DNA damage response favoring tumor progression.

    PubMed

    Seoane, Marcos; Iglesias, Pablo; Gonzalez, Teresa; Dominguez, Fernando; Fraga, Maximo; Aliste, Carlos; Forteza, Jeronimo; Costoya, Jose A

    2008-01-01

    Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRas(V12). Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

  3. The DNA damage response in viral-induced cellular transformation.

    PubMed

    Nikitin, P A; Luftig, M A

    2012-01-31

    The DNA damage response (DDR) has emerged as a critical tumour suppressor pathway responding to cellular DNA replicative stress downstream of aberrant oncogene over-expression. Recent studies have now implicated the DDR as a sensor of oncogenic virus infection. In this review, we discuss the mechanisms by which tumour viruses activate and also suppress the host DDR. The mechanism of tumour virus induction of the DDR is intrinsically linked to the need for these viruses to promote an S-phase environment to replicate their nucleic acid during infection. However, inappropriate expression of viral oncoproteins can also activate the DDR through various mechanisms including replicative stress, direct interaction with DDR components and induction of reactive oxygen species. Given the growth-suppressive consequences of activating the DDR, tumour viruses have also evolved mechanisms to attenuate these pathways. Aberrant expression of viral oncoproteins may therefore promote tumourigenesis through increased somatic mutation and aneuploidy due to DDR inactivation. This review will focus on the interplay between oncogenic viruses and the DDR with respect to cellular checkpoint control and transformation.

  4. WWOX modulates the ATR-mediated DNA damage checkpoint response.

    PubMed

    Abu-Odeh, Mohammad; Hereema, Nyla A; Aqeilan, Rami I

    2016-01-26

    For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability.

  5. WWOX modulates the ATR-mediated DNA damage checkpoint response

    PubMed Central

    Abu-Odeh, Mohammad; Hereema, Nyla A.; Aqeilan, Rami I.

    2016-01-01

    For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability. PMID:26675548

  6. 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.

  7. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    PubMed

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.

  8. 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

  9. Function of ZFAND3 in the DNA Damage Response

    DTIC Science & Technology

    2013-06-01

    to DNA damaging agents . The replication stress reagent hydroxyurea (HU) stalls replication forks, and NS TEX27_1...survival during the replication stress observed in these conditions. The role of ZFAND3 following a variety of damaging agents was examined as proposed in...the procedure effectively captures a maturing chromatin segment of constant length (Fig. 1D). However, PCNA and CAF-1 levels purified with the EdU

  10. Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage.

    PubMed

    Fu, Dechen; Dudimah, Fred Duafalia; Zhang, Jun; Pickering, Anna; Paneerselvam, Jayabal; Palrasu, Manikandan; Wang, Hong; Fei, Peiwen

    2013-03-01

    How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.

  11. 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.

  12. Ku80-deletion suppresses spontaneous tumors and induces a p53-mediated DNA damage response

    PubMed Central

    Holcomb, Valerie B.; Rodier, Francis; Choi, Yong Jun; Busuttil, Rita A.; Vogel, Hannes; Vijg, Jan; Campisi, Judith; Hasty, Paul

    2014-01-01

    Ku80 facilitates DNA repair and therefore should suppress cancer. However, ku80−/− mice exhibit reduced cancer, although they age prematurely and have a shortened life span. We tested the hypothesis that Ku80 deletion suppresses cancer by enhancing cellular tumor suppressive responses to inefficiently repaired DNA damage. In support of this hypothesis, Ku80 deletion ameliorated tumor burden in APCMIN mice, and increased a p53-mediated DNA damage response, DNA lesions, and chromosomal rearrangements. Thus, contrary to its assumed role as a caretaker tumor suppressor, Ku80 facilitates tumor growth most likely by dampening baseline cellular DNA damage responses. PMID:19010925

  13. 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.

  14. 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

  15. BRCA1 in the DNA damage response and at telomeres.

    PubMed

    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.

  16. Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response.

    PubMed

    Stephenson, Anthony A; Taggart, David J; Suo, Zucai

    2017-04-13

    Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

  17. Modulation of inflammation and disease tolerance by DNA damage response pathways.

    PubMed

    Neves-Costa, Ana; Moita, Luis F

    2016-09-30

    The accurate replication and repair of DNA is central to organismal survival. This process is challenged by the many factors that can change genetic information such as replication errors and direct damage to the DNA molecule by chemical and physical agents. DNA damage can also result from microorganism invasion as an integral step of their life cycle or as collateral damage from host defense mechanisms against pathogens. Here we review the complex crosstalk of DNA damage response and immune response pathways that might be evolutionarily connected and argue that DNA damage response pathways can be explored therapeutically to induce disease tolerance through the activation of tissue damage control processes. Such approach may constitute the missing pillar in the treatment of critical illnesses caused by multiple organ failure, such as sepsis and septic shock.

  18. The Role of the DNA Damage Response throughout the Papillomavirus Life Cycle

    PubMed Central

    McKinney, Caleb C.; Hussmann, Katherine L.; McBride, Alison A.

    2015-01-01

    The DNA damage response (DDR) maintains genomic integrity through an elaborate network of signaling pathways that sense DNA damage and recruit effector factors to repair damaged DNA. DDR signaling pathways are usurped and manipulated by the replication programs of many viruses. Here, we review the papillomavirus (PV) life cycle, highlighting current knowledge of how PVs recruit and engage the DDR to facilitate productive infection. PMID:26008695

  19. 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.

  20. Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress

    PubMed Central

    Sorrell, Melanie; Berman, Zachary

    2014-01-01

    To maintain genome stability, cells have evolved various DNA repair pathways to deal with oxidative DNA damage. DNA damage response (DDR) pathways, including ATM-Chk2 and ATR-Chk1 checkpoints, are also activated in oxidative stress to coordinate DNA repair, cell cycle progression, transcription, apoptosis, and senescence. Several studies demonstrate that DDR pathways can regulate DNA repair pathways. On the other hand, accumulating evidence suggests that DNA repair pathways may modulate DDR pathway activation as well. In this review, we summarize our current understanding of how various DNA repair and DDR pathways are activated in response to oxidative DNA damage primarily from studies in eukaryotes. In particular, we analyze the functional interplay between DNA repair and DDR pathways in oxidative stress. A better understanding of cellular response to oxidative stress may provide novel avenues of treating human diseases, such as cancer and neurodegenerative disorders. PMID:24947324

  1. 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

  2. Oxidative DNA Damage Response in Helicobacter pylori-Infected Mongolian Gerbils.

    PubMed

    Bae, Minkyung; Lim, Joo Weon; Kim, Hyeyoung

    2013-09-01

    Helicobacter pylori (H. pylori) induced DNA damage which may be related to gastric cancer development. The DNA damage response coordinates DNA repair, cell-cycle transition, and apoptosis through activation of DNA damage response molecules. The damaged DNA is repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). In the present study, we investigated the changes of HR DNA repair proteins (ataxia-telangiectasia-mutated; ATM, ATM and Rad3-related; ATR), NHEJ repair proteins (Ku70/80), cell cycle regulators (Chk1, Chk2), and apoptosis marker (p53/p-p53) were determined in H. pylori-infected Mongolian gerbils. In addition, the effect of an antioxidant N-acetylcysteine (NAC) on H. pylori-induced DNA damage response was determined to assess the involvement of oxidative stress on DNA damage of the animals infected with H. pylori. One week after intragastric inoculation with H. pylori, Mongolian gerbils were fed with basal diet with or without 3% NAC for 6 weeks. After 6 week, the expression levels of DNA repair proteins (Ku70/80, ATM, ATR), cell cycle regulators (Chk1, Chk2) and apoptosis marker (p-p53/p53) were increased in gastric mucosa of Mongolian gerbils, which was suppressed by NAC treatment. In conclusion, oxidative stress mediates H. pylori-induced DNA damage response including NHEJ and HR repairing processes, cell cycle arrest and apoptosis in gastric mucosa of Mongolian gerbils.

  3. The ATM Kinase Induces MicroRNA Biogenesis in the DNA Damage Response

    PubMed Central

    Zhang, Xinna; Wan, Guohui; Berger, Franklin G.; He, Xiaoming; Lu, Xiongbin

    2011-01-01

    SUMMARY The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one fourth of miRNAs are significantly up-regulated after DNA damage, while loss of ATM abolishes their induction. KSRP (KH-type splicing regulatory protein) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis. PMID:21329876

  4. Dissecting DNA damage response pathways by analyzing protein localization and abundance changes during DNA replication stress

    PubMed Central

    Tkach, Johnny M.; Yimit, Askar; Lee, Anna Y.; Riffle, Michael; Costanzo, Michael; Jaschob, Daniel; Hendry, Jason A.; Ou, Jiongwen; Moffat, Jason; Boone, Charles; Davis, Trisha N.; Nislow, Corey; Brown, Grant W.

    2012-01-01

    Re-localization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein re-organization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by sub-cellular destination allows the identification of pathways that respond to replication stress. We analyzed pairwise combinations of GFP fusions and gene deletion mutants to define and order two novel DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways. PMID:22842922

  5. Interplay between DNA tumor viruses and the host DNA damage response.

    PubMed

    McFadden, Karyn; Luftig, Micah A

    2013-01-01

    Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication.

  6. 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

  7. RNA m(6)A methylation regulates the ultraviolet-induced DNA damage response.

    PubMed

    Xiang, Yang; Laurent, Benoit; Hsu, Chih-Hung; Nachtergaele, Sigrid; Lu, Zhike; Sheng, Wanqiang; Xu, Chuanyun; Chen, Hao; Ouyang, Jian; Wang, Siqing; Ling, Dominic; Hsu, Pang-Hung; Zou, Lee; Jambhekar, Ashwini; He, Chuan; Shi, Yang

    2017-03-23

    Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m(6)A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)(+) transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m(6)A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m(6)A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m(6)A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

  8. Viral DNA Replication-Dependent DNA Damage Response Activation during BK Polyomavirus Infection

    PubMed Central

    Verhalen, Brandy; Justice, Joshua L.; Imperiale, Michael J.

    2015-01-01

    ABSTRACT BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. IMPORTANCE Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR

  9. Choreography of recombination proteins during the DNA damage response.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2009-09-02

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells.

  10. LOX-1, oxidant stress, mtDNA damage, autophagy, and immune response in atherosclerosis.

    PubMed

    Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Dai, Yao; Khaidakov, Magomed; Romeo, Francesco; Mehta, Jawahar L

    2014-07-01

    As a major receptor for oxidized low density lipoprotein (ox-LDL), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is upregulated in many pathophysiological events, including endothelial cell dysfunction and smooth muscle cell growth, as well as monocyte migration and transformation into foam cells, which are present in atherosclerosis and myocardial ischemia. Excessive production of reactive oxygen species (ROS) increases LOX-1 expression, induces mitochondrial DNA damage, and activates autophagy. Damaged mitochondrial DNA that escapes from autophagy induces an inflammatory response. This paper reviews the potential link between LOX-1, mitochondrial DNA damage, autophagy, and immune response in atherosclerosis.

  11. The mef/elf4 transcription factor fine tunes the DNA damage response.

    PubMed

    Sashida, Goro; Bae, Narae; Di Giandomenico, Silvana; Asai, Takashi; Gurvich, Nadia; Bazzoli, Elena; Liu, Yan; Huang, Gang; Zhao, Xinyang; Menendez, Silvia; Nimer, Stephen D

    2011-07-15

    The ATM kinase plays a critical role in initiating the DNA damage response that is triggered by genotoxic stresses capable of inducing DNA double-strand breaks. Here, we show that ELF4/MEF, a member of the ETS family of transcription factors, contributes to the persistence of γH2AX DNA damage foci and promotes the DNA damage response leading to the induction of apoptosis. Conversely, the absence of ELF4 promotes the faster repair of damaged DNA and more rapid disappearance of γH2AX foci in response to γ-irradiation, leading to a radio-resistant phenotype despite normal ATM phosphorylation. Following γ-irradiation, ATM phosphorylates ELF4, leading to its degradation; a mutant form of ELF4 that cannot be phosphorylated by ATM persists following γ-irradiation, delaying the resolution of γH2AX foci and triggering an excessive DNA damage response. Thus, although ELF4 promotes the phosphorylation of H2AX by ATM, its activity must be dampened by ATM-dependent phosphorylation and degradation to avoid an excessive DNA damage response.

  12. Fluorescent sensors of PARP-1 structural dynamics and allosteric regulation in response to DNA damage

    PubMed Central

    Steffen, Jamin D.; McCauley, Michael M.; Pascal, John M.

    2016-01-01

    Poly(ADP-ribose) (PAR) is a posttranslational modification predominantly synthesized by PAR polymerase-1 (PARP-1) in genome maintenance. PARP-1 detects DNA damage, and damage detection is coupled to a massive increase PAR production, primarily attached to PARP-1 (automodification). Automodified PARP-1 then recruits repair factors to DNA damage sites. PARP-1 automodification eventually leads to release from DNA damage thus turning off catalytic activity, although the effects of PAR on PARP-1 structure are poorly understood. The multiple domains of PARP-1 are organized upon detecting DNA damage, creating a network of domain contacts that imposes a major conformational transition in the catalytic domain that increases PAR production. Presented here are two novel fluorescent sensors that monitor the global and local structural transitions of PARP-1 that are associated with DNA damage detection and catalytic activation. These sensors display real-time monitoring of PARP-1 structural transitions upon DNA damage detection, and their reversal upon PARP-1 automodification. The fluorescent sensors are further used to investigate intramolecular and intermolecular PARP-1 activation, followed by the observation that intramolecular activation of PARP-1 is the predominant response to DNA strand breaks in cells. These results provide a unique perspective on the interplay between PARP-1 DNA damage recognition, allosteric regulation, and catalytic activity. PMID:27530425

  13. Fluorescent sensors of PARP-1 structural dynamics and allosteric regulation in response to DNA damage.

    PubMed

    Steffen, Jamin D; McCauley, Michael M; Pascal, John M

    2016-11-16

    Poly(ADP-ribose) (PAR) is a posttranslational modification predominantly synthesized by PAR polymerase-1 (PARP-1) in genome maintenance. PARP-1 detects DNA damage, and damage detection is coupled to a massive increase PAR production, primarily attached to PARP-1 (automodification). Automodified PARP-1 then recruits repair factors to DNA damage sites. PARP-1 automodification eventually leads to release from DNA damage thus turning off catalytic activity, although the effects of PAR on PARP-1 structure are poorly understood. The multiple domains of PARP-1 are organized upon detecting DNA damage, creating a network of domain contacts that imposes a major conformational transition in the catalytic domain that increases PAR production. Presented here are two novel fluorescent sensors that monitor the global and local structural transitions of PARP-1 that are associated with DNA damage detection and catalytic activation. These sensors display real-time monitoring of PARP-1 structural transitions upon DNA damage detection, and their reversal upon PARP-1 automodification. The fluorescent sensors are further used to investigate intramolecular and intermolecular PARP-1 activation, followed by the observation that intramolecular activation of PARP-1 is the predominant response to DNA strand breaks in cells. These results provide a unique perspective on the interplay between PARP-1 DNA damage recognition, allosteric regulation, and catalytic activity.

  14. An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR, DNA DAMAGE CHECKPOINT, AND APOPTOSIS.

    PubMed

    Choi, Jun-Hyuk; Kim, So-Young; Kim, Sook-Kyung; Kemp, Michael G; Sancar, Aziz

    2015-11-27

    DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. ARRB1 enhances the chemosensitivity of lung cancer through the mediation of DNA damage response

    PubMed Central

    Shen, Hongchang; Wang, Liguang; Zhang, Jiangang; Dong, Wei; Zhang, Tiehong; Ni, Yang; Cao, Hongxin; Wang, Kai; Li, Yun; Wang, Yibing; Du, Jiajun

    2017-01-01

    ARRB1 (also known as β-arrestin-1) serves as a multifunctional adaptor contributing to the regulation of signaling pathways. ARRB1 may be involved in DNA damage accumulation; however the underlying mechanism involved is unclear. In the present study, non-small cell lung cancer (NSCLC) cell lines (H520 and SK-MES-1) were transfected with ARRB1 plasmids or small interfering ribonucleic acid (siRNA) and received treatment with DNA-damaging agents (cisplatin and etoposide). A mouse xenograft model was used to assess the impact of ARRB1 on the efficacy of cisplatin in vivo. A total of 30 surgically resected NSCLC patients were recruited for the present study and qRT-PCR was performed to determine the mRNA levels in cancer tissues compared with para-carcinoma tissues. Our data showed that DNA damage was abrogated in the ARRB1-knockdown cells and enhanced in the ARRB1-overexpressing cells. ATR and Chk1 were more activated in the ARRB1-overexpressing cells compared to the ARRB1-knockdown cells, followed by increased H2AX phosphorylation. DNA damage and apoptosis were increased in the ARRB1-overexpressing cells treated with cisplatin. These data provided strong evidence that ARRB1 contributes to the response of NSCLC to DNA-damaging agents and is essential for DNA damage response (DDR). ARRB1 may enhance the efficacy of DNA-damaging agents in NSCLC. PMID:28035404

  20. Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

    PubMed Central

    Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

    2016-01-01

    The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

  1. Histone H3 lysine 56 acetylation and the response to DNA replication fork damage.

    PubMed

    Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien; St-Hilaire, Edlie; Lee, Eun-Hye; Tsao, Sarah; Dorn, Jonas; Maddox, Paul; Lisby, Michael; Pasero, Philippe; Verreault, Alain

    2012-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.

  2. Histone H3 Lysine 56 Acetylation and the Response to DNA Replication Fork Damage

    PubMed Central

    Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien; St-Hilaire, Edlie; Lee, Eun-Hye; Tsao, Sarah; Dorn, Jonas; Maddox, Paul; Lisby, Michael; Pasero, Philippe

    2012-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication. PMID:22025679

  3. Ku70 Serine 155 mediates Aurora B inhibition and activation of the DNA damage response

    PubMed Central

    Fell, Victoria L.; Walden, Elizabeth A.; Hoffer, Sarah M.; Rogers, Stephanie R.; Aitken, Amelia S.; Salemi, Louisa M.; Schild-Poulter, Caroline

    2016-01-01

    The Ku heterodimer (Ku70/Ku80) is the central DNA binding component of the classical non-homologous end joining (NHEJ) pathway that repairs DNA double-stranded breaks (DSBs), serving as the scaffold for the formation of the NHEJ complex. Here we show that Ku70 is phosphorylated on Serine 155 in response to DNA damage. Expression of Ku70 bearing a S155 phosphomimetic substitution (Ku70 S155D) in Ku70-deficient mouse embryonic fibroblasts (MEFs) triggered cell cycle arrest at multiple checkpoints and altered expression of several cell cycle regulators in absence of DNA damage. Cells expressing Ku70 S155D exhibited a constitutive DNA damage response, including ATM activation, H2AX phosphorylation and 53BP1 foci formation. Ku70 S155D was found to interact with Aurora B and to have an inhibitory effect on Aurora B kinase activity. Lastly, we demonstrate that Ku and Aurora B interact following ionizing radiation treatment and that Aurora B inhibition in response to DNA damage is dependent upon Ku70 S155 phosphorylation. This uncovers a new pathway where Ku may relay signaling to Aurora B to enforce cell cycle arrest in response to DNA damage. PMID:27849008

  4. CTCF participates in DNA damage response via poly(ADP-ribosyl)ation

    PubMed Central

    Han, Deqiang; Chen, Qian; Shi, Jiazhong; Zhang, Feng; Yu, Xiaochun

    2017-01-01

    CCCTC-binding factor (CTCF) plays an essential role in regulating the structure of chromatin by binding DNA strands for defining the boundary between active and heterochromatic DNA. However, the role of CTCF in DNA damage response remains elusive. Here, we show that CTCF is quickly recruited to the sites of DNA damage. The fast recruitment is mediated by the zinc finger domain and poly (ADP-ribose) (PAR). Further analyses show that only three zinc finger motifs are essential for PAR recognition. Moreover, CTCF-deficient cells are hypersensitive to genotoxic stress such as ionizing radiation (IR). Taken together, these results suggest that CTCF participate in DNA damage response via poly(ADP-ribosylation). PMID:28262757

  5. DNA damage response induces structural alterations in histone H3–H4

    PubMed Central

    Izumi, Yudai; Fujii, Kentaro; Yamamoto, Satoshi; Matsuo, Koichi; Namatame, Hirofumi; Taniguchi, Masaki; Yokoya, Akinari

    2017-01-01

    Synchrotron-radiation circular-dichroism spectroscopy was used to reveal that the DNA damage response induces a decrement of α-helix and an increment of β-strand contents of histone H3–H4 extracted from X-ray–irradiated human HeLa cells. The trend of the structural alteration was qualitatively opposite to that of our previously reported results for histone H2A–H2B. These results strongly suggest that histones share roles in DNA damage responses, particularly in DNA repair processes and chromatin remodeling, via a specific structural alteration of each histone. PMID:27672100

  6. LKB1 is a DNA damage response protein that regulates cellular sensitivity to PARP inhibitors

    PubMed Central

    Wang, Huibo; Wang, Shuai; Hang, Wei; Zeng, Qinghua; Quan, Cheng-Shi; Zhai, Ying-Xian; Wang, Jian-Wei; Shen, Xiang-Feng; Jian, Yong-Ping; Zhao, Rui-Xun; Werle, Kaitlin D.; Cui, Rutao; Liang, Jiyong; Li, Yu-Lin; Xu, Zhi-Xiang

    2016-01-01

    Liver kinase B1 (LKB1) functions as a tumor suppressor encoded by STK11, a gene that mutated in Peutz-Jeghers syndrome and in sporadic cancers. Previous studies showed that LKB1 participates in IR- and ROS-induced DNA damage response (DDR). However, the impact of LKB1 mutations on targeted cancer therapy remains unknown. Herein, we demonstrated that LKB1 formed DNA damage-induced nuclear foci and co-localized with ataxia telangiectasia mutated kinase (ATM), γ-H2AX, and breast cancer susceptibility 1 (BRCA1). ATM mediated LKB1 phosphorylation at Thr 363 following the exposure of cells to ionizing radiation (IR). LKB1 interacted with BRCA1, a downstream effector in DDR that is recruited to sites of DNA damage and functions directly in homologous recombination (HR) DNA repair. LKB1 deficient cells exhibited delayed DNA repair due to insufficient HR. Notably, LKB1 deficiency sensitized cells to poly (ADP-ribose) polymerase (PARP) inhibitors. Thus, we have demonstrated a novel function of LKB1 in DNA damage response. Cancer cells lacking LKB1 are more susceptible to DNA damage-based therapy and, in particular, to drugs that further impair DNA repair, such as PARP inhibitors. PMID:27705915

  7. 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.

  8. 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

  9. Posttranscriptional regulation of gene expression-adding another layer of complexity to the DNA damage response.

    PubMed

    Boucas, Jorge; Riabinska, Arina; Jokic, Mladen; Herter-Sprie, Grit S; Chen, Shuhua; Höpker, Katja; Reinhardt, H Christian

    2012-01-01

    In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR), is primarily thought to consist of two components-a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs) in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs) still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RBPs and the control of their function through DNA damage-activated protein kinases.

  10. Chronic inflammation-related DNA damage response: a driving force of gastric cardia carcinogenesis.

    PubMed

    Lin, Runhua; Xiao, Dejun; Guo, Yi; Tian, Dongping; Yun, Hailong; Chen, Donglin; Su, Min

    2015-02-20

    Gastric cardia cancer (GCC) is a highly aggressive disease associated with chronic inflammation. To investigate the relationship between DNA damage response (DDR) and chronic inflammation, we collected 100 non-tumor gastric cardia specimens of Chaoshan littoral, a high-risk region for esophageal and gastric cardia cancer. A significantly higher proportion of severe chronic inflammation was found in dysplastic epithelia (80.9%) in comparison with that in non-dysplastic tissues (40.7%) (P<0.001). Immunohistochemical analysis demonstrated that DNA damage response was parallel with the chronic inflammation degrees from normal to severe inflammation (P<0.05). We found that DNA damage response was progressively increased with the progression of precancerous lesions (P<0.05). These findings provide pathological evidence that persistent chronic inflammation-related DNA damage response may be a driving force of gastric cardia carcinogenesis. Based on these findings, DNA damage response in non-malignant tissues may become a promising biomedical marker for predicting malignant transformation in the gastric cardia.

  11. Cross-talk between carbon metabolism and the DNA damage response in S. cerevisiae

    PubMed Central

    Simpson-Lavy, Kobi J.; Bronstein, Alex; Kupiec, Martin; Johnston, Mark

    2015-01-01

    Yeast cells with DNA damage avoid respiration, presumably because products of oxidative metabolism can be harmful to DNA. We show that DNA damage inhibits the activity of the Snf1 (AMP-activated) protein kinase (AMPK), which activates expression of genes required for respiration. Glucose and DNA damage upregulate SUMOylation of Snf1, catalyzed by the SUMO E3-ligase Mms21, which inhibits SNF1 activity. The DNA damage checkpoint kinases Mec1/ATR and Tel1/ATM, as well as the nutrient sensing protein kinase A (PKA), regulate Mms21 activity towards Snf1. Mec1 and Tel1 are required for two SNF1-regulated processes—glucose sensing and ADH2 gene expression—even without exogenous genotoxic stress. Our results imply that inhibition of Snf1 by SUMOylation is a mechanism by which cells lower their respiration in response to DNA damage. This raises the possibility that activation of DNA damage checkpoint mechanisms could contribute to aerobic fermentation (Warburg effect), a hallmark of cancer cells. PMID:26344768

  12. Oleandrin induces DNA damage responses in cancer cells by suppressing the expression of Rad51

    PubMed Central

    Bao, Zhengqiang; Tian, Baoping; Wang, Xiaohui; Feng, Hanrong; Liang, Ye; Chen, Zhihua; Li, Wen; Shen, Huahao; Ying, Songmin

    2016-01-01

    Oleandrin is a monomeric compound extracted from leaves and seeds of Nerium oleander. It had been reported that oleandrin could effectively inhibit the growth of human cancer cells. However, the specific mechanisms of the oleandrin-induced anti-tumor effects remain largely unclear. Genomic instability is one of the main features of cancer cells, it can be the combined effect of DNA damage and tumour-specific DNA repair defects. DNA damage plays important roles during tumorigenesis. In fact, most of the current chemotherapy agents were designed to kill cancer cells by inducing DNA damage. In this study, we found that oleandrin was effective to induce apoptosis in cancer cells, and cause rapid DNA damage response, represented by nuclear RPA (Replication Protein A, a single strand DNA binding protein) and γH2AX(a marker for DNA double strand breaks) foci formation. Interestingly, expression of RAD51, a key protein involved in homologous recombination (HR), was suppressed while XRCC1 was up-regulated in oleandrin treated cancer cells. These results suggested that XRCC1 may play a predominant role in repairing oleandrin-induced DNA damage. Collectively, oleandrin may be a potential anti-tumor agent by suppressing the expression of Rad51. PMID:27449097

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

    PubMed

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

    2016-04-05

    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.

  14. TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

    PubMed

    Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

    2016-01-01

    DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

  15. A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.

    PubMed Central

    Sebastian, J; Sancar, G B

    1991-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription. Images PMID:1763039

  16. Regulation of DNA damage responses and cell cycle progression by hMOB2

    PubMed Central

    Gomez, Valenti; Gundogdu, Ramazan; Gomez, Marta; Hoa, Lily; Panchal, Neelam; O’Driscoll, Mark; Hergovich, Alexander

    2014-01-01

    Mps one binder proteins (MOBs) are conserved regulators of essential signalling pathways. Biochemically, human MOB2 (hMOB2) can inhibit NDR kinases by competing with hMOB1 for binding to NDRs. However, biological roles of hMOB2 have remained enigmatic. Here, we describe novel functions of hMOB2 in the DNA damage response (DDR) and cell cycle regulation. hMOB2 promotes DDR signalling, cell survival and cell cycle arrest after exogenously induced DNA damage. Under normal growth conditions in the absence of exogenously induced DNA damage hMOB2 plays a role in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest. Unexpectedly, these molecular and cellular phenotypes are not observed upon NDR manipulations, indicating that hMOB2 performs these functions independent of NDR signalling. Thus, to gain mechanistic insight, we screened for novel binding partners of hMOB2, revealing that hMOB2 interacts with RAD50, facilitating the recruitment of the MRE11-RAD50-NBS1 (MRN) DNA damage sensor complex and activated ATM to DNA damaged chromatin. Taken together, we conclude that hMOB2 supports the DDR and cell cycle progression. PMID:25460043

  17. 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

  18. 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

  19. Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response.

    PubMed

    Salton, Maayan; Lerenthal, Yaniv; Wang, Shih-Ya; Chen, David J; Shiloh, Yosef

    2010-04-15

    The DNA damage response (DDR) is a complex signaling network that is induced by DNA lesions and vigorously activated by double strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which phosphorylates key players in its various branches. SFPQ (PSF) and NONO (p54) are nuclear proteins that interact with each other and have diverse roles in nucleic acids metabolism. The SFPQ/NONO heterodimer was previously found to enhance DNA strand break rejoining in vitro. Our attention was drawn to these two proteins as they interact with the nuclear matrix protein Matrin 3 (MATR3), which we found to be a novel ATM target. We asked whether SFPQ and NONO too are involved in the DSB response. Proteins that function at the early phase of this response are often recruited to the damaged sites. We observed rapid recruitment of SFPQ/NONO to sites of DNA damage induced by laser microbeam. In MATR3 knockdown cells SFPQ/NONO retention at DNA damage sites was prolonged. SFPQ and MATR3 depletion led to abnormal accumulation of cells at the S-phase of the cell cycle following treatment with the radiomimetic chemical neocarzinostatin. Notably, proteins involved in DSB repair via nonhomologous end-joining co-immunoprecipitated with NONO; SFPQ depletion delayed DSB repair. Collectively the data suggest that SFPQ, NONO and MATR3 are involved in the early stage of the DSB response, setting the scene for DSB repair.

  20. Epigenetic and genetic factors in the cellular response to radiations and DNA-damaging chemicals

    SciTech Connect

    Williams, J.R.; D'Arpa, P.

    1981-07-01

    DNA-damaging agents are widely used as therapeutic tools for a variety of disease states. Many such agents are considered to produce detrimental side effects. Thus, it is important to evaluate both therapeutic efficacy and potential risk. DNA-damaging agents can be so evaluated by comparison to agents whose therapeutic benefit and potential hazards are better known. We propose a framework for such comparison, demonstrating that a simple transformation of cytotoxicity-dose response patterns permits a facile comparison of variation between cells exposed to a single DNA-damaging agent or to different cytotoxic agents. Further, by transforming data from experiments which compare responses of 2 cell populations to an effects ratio, different patterns for the changes in cytotoxicity produced by epigenetic and genetic factors were compared. Using these transformations, we found that there is a wide variation (a factor of 4) between laboratories for a single agent (UVC) and only a slightly larger variation (factor of 6) between normal cell response for different types of DNA-damaging agents (x-ray, UVC, alkylating agents, crosslinking agents). Epigenetic factors such as repair and recovery appear to be a factor only at higher dose levels. Comparison in the cytotoxic effect of a spectrum of DNA-damaging agents in xeroderma pigmentosum, ataxia telangiectasia, and Fanconi's anemia cells indicates significantly different patterns, implying that the effect, and perhaps the nature, of these genetic conditions are quite different.

  1. 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.

  2. Dose-response relationship for rat liver DNA damage caused by 1,2-dimethylhydrazine.

    PubMed

    Kitchin, K T; Brown, J L

    1996-12-02

    An experimental approach was taken to the question of dose-response curves for chemical carcinogenesis, using DNA damage as a biomarker. Female rats were give 13 different doses of 1,2-dimethylhydrazine (from 1.4 to 135,000 micrograms/kg) and the subsequent hepatic DNA damage was determined by the alkaline elution technique. DMH doses below 450 micrograms/kg did not significantly damage DNA; all DMH doses of 1000 micrograms/kg or higher damaged rat hepatic DNA (P < 0.05). In this study the x values (dose) ranged over five orders of magnitude and the y values (DNA damage) ranged 30-fold. Ten different regression models (linear, quadratic, cubic, power, and six nonlinear transition models) were compared in their ability to fit the experimental data. With respect to log transformed dose, the six nonlinear transition equations fit the data considerably better than the four power type of equations. A sigmoid model fit to the log transformed dose of 1,2-dimethylhydrazine had an r2 of 0.9979, a degree of freedom adjusted r2 of 0.9969, a F-statistic of 1,457, and a fit standard error of 0.50. With respect to untransformed dose, only three equations (sigmoid, cascade and gaussian cumulative) could creditably fit the DMH data. The experimental results are interpreted with respect to hormesis, use of log transformed dose, sigmoid dose-response models, thresholds of biological response and cancer risk assessment.

  3. 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

  4. Induction of a Cellular DNA Damage Response by Porcine Circovirus Type 2 Facilitates Viral Replication and Mediates Apoptotic Responses

    PubMed Central

    Wei, Li; Zhu, Shanshan; Wang, Jing; Quan, Rong; Yan, Xu; Li, Zixue; Hou, Lei; Wang, Naidong; Yang, Yi; Jiang, Haijun; Liu, Jue

    2016-01-01

    Cellular DNA damage response (DDR) triggered by infection of DNA viruses mediate cell cycle checkpoint activation, DNA repair, or apoptosis induction. In the present study, infection of porcine circovirus type 2 (PCV2), which serves as a major etiological agent of PCV2-associated diseases (PCVAD), was found to elicit a DNA damage response (DDR) as observed by the phosphorylation of H2AX and RPA32 following infection. The response requires active viral replication, and all the ATM (ataxia telangiectasia-mutated kinase), ATR (ATM- and Rad3-related kinase), and DNA-PK (DNA-dependent protein kinase) are the transducers of the DDR signaling events in the PCV2-infected cells as demonstrated by the phosphorylation of ATM, ATR, and DNA-PK signalings as well as reductions in their activations after treatment with specific kinase inhibitors. Inhibitions of ATM, ATR, and DNA-PK activations block viral replication and prevent apoptotic responses as observed by decreases in cleaved poly-ADP ribose polymerase (PARP) and caspase-3 as well as fragmented DNA following PCV2 infection. These results reveal that PCV2 is able to exploit the cellular DNA damage response machinery for its own efficient replication and for apoptosis induction, further extending our understanding for the molecular mechanism of PCV2 infection. PMID:27982097

  5. Potentiated DNA Damage Response in Circulating Breast Tumor Cells Confers Resistance to Chemotherapy*

    PubMed Central

    Gong, Chang; Liu, Bodu; Yao, Yandan; Qu, Shaohua; Luo, Wei; Tan, Weige; Liu, Qiang; Yao, Herui; Zou, Lee; Su, Fengxi; Song, Erwei

    2015-01-01

    Circulating tumor cells (CTCs) are seeds for cancer metastasis and are predictive of poor prognosis in breast cancer patients. Whether CTCs and primary tumor cells (PTCs) respond to chemotherapy differently is not known. Here, we show that CTCs of breast cancer are more resistant to chemotherapy than PTCs because of potentiated DNA repair. Surprisingly, the chemoresistance of CTCs was recapitulated in PTCs when they were detached from the extracellular matrix. Detachment of PTCs increased the levels of reactive oxygen species and partially activated the DNA damage checkpoint, converting PTCs to a CTC-like state. Inhibition of checkpoint kinases Chk1 and Chk2 in CTCs reduces the basal checkpoint response and sensitizes CTCs to DNA damage in vitro and in mouse xenografts. Our results suggest that DNA damage checkpoint inhibitors may benefit the chemotherapy of breast cancer patients by suppressing the chemoresistance of CTCs and reducing the risk of cancer metastasis. PMID:25897074

  6. Potentiated DNA Damage Response in Circulating Breast Tumor Cells Confers Resistance to Chemotherapy.

    PubMed

    Gong, Chang; Liu, Bodu; Yao, Yandan; Qu, Shaohua; Luo, Wei; Tan, Weige; Liu, Qiang; Yao, Herui; Zou, Lee; Su, Fengxi; Song, Erwei

    2015-06-12

    Circulating tumor cells (CTCs) are seeds for cancer metastasis and are predictive of poor prognosis in breast cancer patients. Whether CTCs and primary tumor cells (PTCs) respond to chemotherapy differently is not known. Here, we show that CTCs of breast cancer are more resistant to chemotherapy than PTCs because of potentiated DNA repair. Surprisingly, the chemoresistance of CTCs was recapitulated in PTCs when they were detached from the extracellular matrix. Detachment of PTCs increased the levels of reactive oxygen species and partially activated the DNA damage checkpoint, converting PTCs to a CTC-like state. Inhibition of checkpoint kinases Chk1 and Chk2 in CTCs reduces the basal checkpoint response and sensitizes CTCs to DNA damage in vitro and in mouse xenografts. Our results suggest that DNA damage checkpoint inhibitors may benefit the chemotherapy of breast cancer patients by suppressing the chemoresistance of CTCs and reducing the risk of cancer metastasis.

  7. 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

  8. The Role of Mms22p in DNA Damage Response in Candida albicans

    PubMed Central

    Yan, Lan; Xiong, Juan; Lu, Hui; Lv, Quan-zhen; Ma, Qian-yao; Côte, Pierre; Whiteway, Malcolm; Jiang, Yuan-ying

    2015-01-01

    To ensure correct DNA replication, eukaryotes have signaling pathways that respond to replication-associated DNA damage and trigger repair. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, a complex of proteins, including the cullin protein Rtt101p and two adapter proteins Mms22p and Mms1p, is important for proper response to replication stress. We have investigated this system in Candida albicans. In this pathogen, Mms22p is important for recovery from DNA replication damage induced by agents including methylmethane sulfonate, camptothecin, and ionizing radiation. Although no clear ortholog of Mms1p has been identified in C. albicans, loss of either Mms22p or Rtt101p generates similar damage sensitivity, consistent with a common function. In S. cerevisiae, the Mrc1p−Csm3p−Tof1p complex stabilizes stalled replication forks and activates a replication checkpoint and interacts with Mms22p. A similar complex in S. pombe, consisting of the Tof1p and Csm3p orthologs Swi1p and Swi3p, along with the fission yeast Mrc1p, genetically also interacts with Mms22p. Intriguingly in C. albicans only Mrc1p and Csm3p appear involved in damage repair, and Mms22p is required for responding to DNA damage agents in MRC1 or CSM3 conditional mutants. In C. albicans, although the loss of RAD57 greatly impairs response in the pathogen to many DNA-damaging agents, lethality due to camptothecin damage requires concomitant loss of Rad57p and Mms22p, suggesting that Mms22p is only essential for homologous recombination induced by camptothecin. These results establish that although C. albicans uses conserved cellular modules to respond to DNA damage and replication blocks, the specific details of these modules differ significantly from the S. cerevisiae model. PMID:26438292

  9. Function of ZFAND3 in the DNA Damage Response

    DTIC Science & Technology

    2011-06-01

    replication fork, such as polymerase epsilon. Therefore, iPOND provides improved sensitivity of detection for replisome components compared to the...enrichment of DSB repair proteins Mre11, KU70/80 and the recombinase Rad51 at stalled forks. Our data suggest that persistently stalled forks collapse...approach: Poly(ADP) Ribose Polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. J Clin Oncol 26(22

  10. 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

    2015-06-24

    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.

  11. 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.

  12. The DNA-damage response: new molecular insights and new approaches to cancer therapy

    PubMed Central

    Jackson, Stephen P.

    2010-01-01

    The DNA of all cells is continually under assault from a wide range of DNA-damaging agents. To counter this threat to their genetic integrity, cells possess systems, collectively known as the DNA-damage response (DDR), to detect DNA damage, signal its presence and mediate its repair. Here, I provide an overview of the DDR and then describe how work in my laboratory and elsewhere has identified some of the key protein players that mediate cellular responses to the most cytotoxic form of DNA damage: the DNA double-strand break (DSB). Next, I discuss some of my laboratory’s recent work, which has revealed that the way cells respond to DSBs is modulated in a cell-cycle dependent manner to ensure that the cell uses the DSB repair system that is most suited to its cell-cycle stage. Finally, I explain how our increasing knowledge of the DDR is suggesting new avenues for treating cancer and provide an example of a DDR inhibitory drug that is showing promise in clinical trials. PMID:19442242

  13. 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

  14. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  15. 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

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

    PubMed Central

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

    2016-01-01

    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

  17. DNA breakage and induction of DNA damage response proteins precede the appearance of visible mutant huntingtin aggregates.

    PubMed

    Illuzzi, Jennifer; Yerkes, Sarah; Parekh-Olmedo, Hetal; Kmiec, Eric B

    2009-02-15

    Huntington's disease (HD) is a neurodegenerative disorder that follows an autosomal-dominant inheritance pattern. The pathogenesis of the disease depends on the degree of expansion of triplet (CAG) repeats located in the first exon on the gene. An expanded polyglutamine tract within the protein huntingtin (Htt) enables a gain-of-function phenotype that is often exhibited by a dysfunctional oligomerization process and the formation of protein aggregates. How this process leads to neurodegeneration remains undefined. We report that expression of a Htt-fragment containing an expanded glutamine tract induces DNA damage and activates the DNA damage response pathway. Both single-strand and double-strand breaks are observed as the mutant protein accumulates in the cell; these breaks precede the appearance of detectable protein aggregates containing mutant Htt. We also observe activation of H2AX, ATM, and p53 in cells expressing mutant Htt, a predictable response in cells containing chromosomal breakage. Expression of wild-type Htt does not affect the integrity of DNA, nor does it activate the same pathway. Furthermore, DNA damage and activated H2AX are present in HD transgenic mice before the formation of mutant Htt aggregates and HD pathogenesis. Taken together, our data suggest that the expression of mutant Htt causes an accumulation of DNA breaks that activates the DNA damage response pathway, a process that can disable cell function. Because these events can lead to apoptosis, it is possible that the DNA damage response pathway activated by single- and double-strand breaks that we found contributes to neurodegeneration.

  18. 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.

  19. Interplay between histone H3 lysine 56 deacetylation and chromatin modifiers in response to DNA damage.

    PubMed

    Simoneau, Antoine; Delgoshaie, Neda; Celic, Ivana; Dai, Junbiao; Abshiru, Nebiyu; Costantino, Santiago; Thibault, Pierre; Boeke, Jef D; Verreault, Alain; Wurtele, Hugo

    2015-05-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage-induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage-induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage.

  20. Bidirectional coupling of splicing and ATM signaling in response to transcription-blocking DNA damage

    PubMed Central

    Tresini, Maria; Marteijn, Jurgen A.; Vermeulen, Wim

    2016-01-01

    ABSTRACT In response to DNA damage cells activate intricate protein networks to ensure genomic fidelity and tissue homeostasis. DNA damage response signaling pathways coordinate these networks and determine cellular fates, in part, by modulating RNA metabolism. Here we discuss a replication-independent pathway activated by transcription-blocking DNA lesions, which utilizes the ATM signaling kinase to regulate spliceosome function in a reciprocal manner. We present a model according to which, displacement of co-transcriptional spliceosomes from lesion-arrested RNA polymerases, culminates in R-loop formation and non-canonical ATM activation. ATM signals in a feed-forward fashion to further impede spliceosome organization and regulates UV-induced gene expression and alternative splicing genome-wide. This reciprocal coupling between ATM and the spliceosome highlights the importance of ATM signaling in the cellular response to transcription-blocking lesions and supports a key role of the splicing machinery in this process. PMID:26913497

  1. The actin depolymerizing factor (ADF)/cofilin signaling pathway and DNA damage responses in cancer.

    PubMed

    Chang, Chun-Yuan; Leu, Jyh-Der; Lee, Yi-Jang

    2015-02-13

    The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy.

  2. Polycomb protein EZH2 regulates cancer cell fate decision in response to DNA damage.

    PubMed

    Wu, Z; Lee, S T; Qiao, Y; Li, Z; Lee, P L; Lee, Y J; Jiang, X; Tan, J; Aau, M; Lim, C Z H; Yu, Q

    2011-11-01

    Polycomb protein histone methyltransferase enhancer of Zeste homologe 2 (EZH2) is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating DNA damage response. Here, we show that EZH2 is an important determinant of cell fate decision in response to genotoxic stress. EZH2 depletion results in abrogation of both cell cycle G1 and G2/M checkpoints, directing DNA damage response toward predominant apoptosis in both p53-proficient and p53-deficient cancer cells, but not in normal cells. Mechanistically, EZH2 regulates DNA damage response in p53 wild-type cells mainly through transcriptional repression of FBXO32, which binds to and directs p21 for proteasome-mediated degradation, whereas it affects p53-deficient cells through regulating Chk1 activation by a distinct mechanism. Furthermore, pharmacological depletion of EZH2 phenocopies the effects of EZH2 knockdown on cell cycle checkpoints and apoptosis. These data unravel a crucial role of EZH2 in determining the cancer cell outcome following DNA damage and suggest that therapeutic targeting oncogenic EZH2 might serve as a strategy for improving conventional chemotherapy in a given malignancy.

  3. 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

  4. Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia – breast cancer susceptibility (FA-BRCA) DNA damage response network

    PubMed Central

    Abraham, Jessy; Balbo, Silvia; Crabb, David; Brooks, P.J.

    2011-01-01

    Background We recently reported that exposure of human cells in vitro to acetaldehyde resulted in activation of the Fanconi anemia-breast cancer associated (FA-BRCA) DNA damage response network. Methods To determine whether intracellular generation of acetaldehyde from ethanol metabolism can cause DNA damage and activate the FA-BRCA network, we engineered HeLa cells to metabolize alcohol by expression of human alcohol dehydrogenase 1B. Results Incubation of HeLa-ADH1B cells with ethanol (20 mM) resulted in acetaldehyde accumulation in the media which was prevented by co-incubation with 4-methyl pyrazole (4-MP), a specific inhibitor of ADH. Ethanol treatment of HeLa-ADH1B cells produced a 4-fold increase in the acetaldehyde-DNA adduct, N2-ethylidene-dGuo, and also resulted in activation of the Fanconi anemia -breast cancer susceptibility (FA-BRCA) DNA damage response network, as indicated by a monoubiquitination of FANCD2, and phosphorylation of BRCA1. Ser 1524 was identified as one site of BRCA1 phosphorylation. The increased levels of DNA adducts, FANCD2 monoubiquitination, and BRCA1 phosphorylation were all blocked by 4-MP, indicating that acetaldehyde, rather than ethanol itself, was responsible for all three responses. Importantly, the ethanol concentration we used is within the range that can be attained in the human body during social drinking. Conclusions Our results indicate that intracellular metabolism of ethanol to acetaldehyde results in DNA damage which activates the FA-BRCA DNA damage response network. PMID:21919919

  5. 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

  6. Initiation of the ATM-Chk2 DNA damage response through the base excision repair pathway.

    PubMed

    Chou, Wen-Cheng; Hu, Ling-Yueh; Hsiung, Chia-Ni; Shen, Chen-Yang

    2015-08-01

    The DNA damage response (DDR) is activated by various genotoxic stresses. Base lesions, which are structurally simple and predominantly fixed by base excision repair (BER), can trigger the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway, a DDR component. How these lesions trigger DDR remains unclear. Here we show that, for alkylation damage, methylpurine-DNA glycosylase (MPG) and apurinic/apyrimidinic endonuclease 1, both of which function early in BER, are required for ATM-Chk2-dependent DDR. In addition, other DNA glycosylases, including uracil-DNA glycosylase and 8-oxoguanine glycosylase, which are involved in repairing deaminated bases and oxidative damage, also induced DDR. The early steps of BER therefore play a vital role in modulating the ATM-Chk2 DDR in response to base lesions, facilitating downstream BER processing for repair, in which the formation of a single-strand break was shown to play a critical role. Moreover, MPG knockdown rescued cell lethality, its overexpression led to cell death triggered by DNA damage and, more interestingly, higher MPG expression in breast and ovarian cancers corresponded with a greater probability of relapse-free survival after chemotherapy, underscoring the importance of glycosylase-dependent DDR. This study highlights the crosstalk between BER and DDR that contributes to maintaining genomic integrity and may have clinical applications in cancer therapy.

  7. Controlling the response to DNA damage by the APC/C-Cdh1.

    PubMed

    de Boer, H Rudolf; Guerrero Llobet, S; van Vugt, Marcel A T M

    2016-03-01

    Proper cell cycle progression is safeguarded by the oscillating activities of cyclin/cyclin-dependent kinase complexes. An important player in the regulation of mitotic cyclins is the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. Prior to entry into mitosis, the APC/C remains inactive, which allows the accumulation of mitotic regulators. APC/C activation requires binding to either the Cdc20 or Cdh1 adaptor protein, which sequentially bind the APC/C and facilitate targeting of multiple mitotic regulators for proteasomal destruction, including Securin and Cyclin B, to ensure proper chromosome segregation and mitotic exit. Emerging data have indicated that the APC/C, particularly in association with Cdh1, also functions prior to mitotic entry. Specifically, the APC/C-Cdh1 is activated in response to DNA damage in G2 phase cells. These observations are in line with in vitro and in vivo genetic studies, in which cells lacking Cdh1 expression display various defects, including impaired DNA repair and aberrant cell cycle checkpoints. In this review, we summarize the current literature on APC/C regulation in response to DNA damage, the functions of APC/C-Cdh1 activation upon DNA damage, and speculate how APC/C-Cdh1 can control cell fate in the context of persistent DNA damage.

  8. 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

  9. 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.

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

    PubMed

    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 gammaH2AX, 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.

  11. Elements that Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome

    PubMed Central

    Iyama, Teruaki; Wilson, David M.

    2015-01-01

    Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration, and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts, and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage, and is most rapid and robust as follows: ICLs > DSBs > monoadducts > oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB, and in particular its ubiquitin-binding domain, were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts, but not oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS. PMID:26616585

  12. Genomic instability and DNA damage responses in progeria arising from defective maturation of prelamin A.

    PubMed

    Musich, Phillip R; Zou, Yue

    2009-01-01

    Progeria syndromes have in common a premature aging phenotype and increased genome instability. The susceptibility to DNA damage arises from a compromised repair system, either in the repair proteins themselves or in the DNA damage response pathways. The most severe progerias stem from mutations affecting lamin A production, a filamentous protein of the nuclear lamina. Hutchinson-Gilford progeria syndrome (HGPS) patients are heterozygous for aLMNA gene mutation while Restrictive Dermopathy (RD) individuals have a homozygous deficiency in the processing protease Zmpste24. These mutations generate the mutant lamin A proteins progerin and FC-lamina A, respectively, which cause nuclear deformations and chromatin perturbations. Genome instability is observed even though genome maintenance and repair genes appear normal. The unresolved question is what features of the DNA damage response pathways are deficient in HGPS and RD cells. Here we review and discuss recent findings which resolve some mechanistic details of how the accumulation of progerin/FC-lamin A proteins may disrupt DNA damage response pathways in HGPS and RD cells. As the mutant lamin proteins accumulate they sequester replication and repair factors, leading to stalled replication forks which collapse into DNA double-strand beaks (DSBs). In a reaction unique to HGPS and RD cells these accessible DSB termini bind Xeroderma pigmentosum group A (XPA) protein which excludes normal binding by DNA DSB repair proteins. The bound XPA also signals activation of ATM and ATR, arresting cell cycle progression, leading to arrested growth. In addition, the effective sequestration of XPA at these DSB damage sites makes HGPS and RD cells more sensitive to ultraviolet light and other mutagens normally repaired by the nucleotide excision repair pathway of which XPA is a necessary and specific component.

  13. 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

  14. FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway.

    PubMed

    Ling, Chen; Ishiai, Masamichi; Ali, Abdullah Mahmood; Medhurst, Annette L; Neveling, Kornelia; Kalb, Reinhard; Yan, Zhijiang; Xue, Yutong; Oostra, Anneke B; Auerbach, Arleen D; Hoatlin, Maureen E; Schindler, Detlev; Joenje, Hans; de Winter, Johan P; Takata, Minoru; Meetei, Amom Ruhikanta; Wang, Weidong

    2007-04-18

    The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100-deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA-BRCA DNA damage response network.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. A novel role of the chromokinesin Kif4A in DNA damage response

    PubMed Central

    Wu, Guikai; Zhou, Longen; Khidr, Lily; Guo, Xuning Emily; Kim, Wankee; Lee, Young Mi; Krasieva, Tatiana; Chen, Phang-Lang

    2011-01-01

    Chromokinesins are microtubule-motor molecules that possess chromatin binding activity and are important for mitotic and meiotic regulation. The chromokinesin-member Kif4A is unique in that it localizes to nucleus during interphase of the cell cycle. Kif4 deletion by gene targeting in mouse embryonic cells was known to associate with DNA damage response. However, its precise role in DNA damage or repair pathway is not clear. Here we report that Kif4A associates with BRCA2 in a biochemical identification and that the interaction is mediated by the Kif4A C-terminal cargo-binding domain and BRCA2 C-terminal conserved region. Upon nucleus-specific laser micro-irradiation, Kif4A was rapidly recruited to sites of DNA damage. Significantly, the depletion of Kif4A from cells by shRNA impaired the ionizing-radiation induced foci (IRIF) formation of Rad51, both quantitatively and qualitatively. In contrast, the IRIF of γ-H2AX or NBS1 was largely intact. Moreover, Kif4A knockdown rendered cells hypersensitive to ionizing radiation in a colonogenic survival assay. We further demonstrated that Kif4A deficiency led to significantly decreased homologous recombination in an I-SceI endonuclease induced in vivo recombination assay. Together, our results suggest a novel role for a chromokinesin family member in the DNA damage response by modulating the BRCA2/Rad51 pathway. PMID:18604178

  20. A novel role of the chromokinesin Kif4A in DNA damage response.

    PubMed

    Wu, Guikai; Zhou, Longen; Khidr, Lily; Guo, Xuning Emily; Kim, Wankee; Lee, Young Mi; Krasieva, Tatiana; Chen, Phang-Lang

    2008-07-01

    Chromokinesins are microtubule-motor molecules that possess chromatin binding activity and are important for mitotic and meiotic regulation. The chromokinesin-member Kif4A is unique in that it localizes to nucleus during interphase of the cell cycle. Kif4 deletion by gene targeting in mouse embryonic cells was known to associate with DNA damage response. However, its precise role in DNA damage or repair pathway is not clear. Here we report that Kif4A associates with BRCA2 in a biochemical identification and that the interaction is mediated by the Kif4A C-terminal cargo-binding domain and BRCA2 C-terminal conserved region. Upon nucleus-specific laser micro-irradiation, Kif4A was rapidly recruited to sites of DNA damage. Significantly, the depletion of Kif4A from cells by shRNA impaired the ionizing-radiation induced foci (IRIF) formation of Rad51, both quantitatively and qualitatively. In contrast, the IRIF of gamma-H2AX or NBS1 was largely intact. Moreover, Kif4A knockdown rendered cells hypersensitive to ionizing radiation in a colonogenic survival assay. We further demonstrated that Kif4A deficiency led to significantly decreased homologous recombination in an I-SceI endonuclease induced in vivo recombination assay. Together, our results suggest a novel role for a chromokinesin family member in the DNA damage response by modulating the BRCA2/Rad51 pathway.

  1. Differential chromatin proteomics of the MMS-induced DNA damage response in yeast

    PubMed Central

    2011-01-01

    Background Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins. Results Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress. Conclusion The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response. PMID:21967861

  2. Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

    PubMed Central

    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

  3. Cell-free Xenopus egg extracts for studying DNA damage response pathways.

    PubMed

    Cupello, Steven; Richardson, Christine; Yan, Shan

    2016-01-01

    In response to a variety of DNA replication stress or DNA damaging agents, the DNA damage response (DDR) pathways are triggered for cells to coordinate DNA repair, cell cycle checkpoints, apoptosis, and senescence. Cell-free Xenopus egg extracts, derived from the eggs of African clawed frogs (Xenopus laevis), have been widely used for studies concerning DDR pathways. In this review, we focus on how different experimental systems have been established using Xenopus egg extracts to investigate the DDR pathways that are activated in response to DNA replication stress, double-strand breaks (DSBs), inter-strand crosslinks (ICLs), and oxidative stress. We summarize how molecular details of DDR pathways are dissected by the mechanistic studies with Xenopus egg extracts. We also provide an update on the regulation of translesion DNA synthesis (TLS) polymerases (Pol ĸ and REV1) in the DDR pathways. A better understanding of DDR pathways using Xenopus egg extracts has opened new avenues for future cancer therapeutics. Finally, we offer our perspectives of future directions for studies of DDR pathways with Xenopus egg extracts.

  4. 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

  5. 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

  6. DNA damage response and Ku80 function in the vertebrate embryo

    PubMed Central

    Bladen, Catherine L.; Lam, Wai K.; Dynan, William S.; Kozlowski, David J.

    2005-01-01

    Cellular responses to DNA damage reflect the dynamic integration of cell cycle control, cell–cell interactions and tissue-specific patterns of gene regulation that occurs in vivo but is not recapitulated in cell culture models. Here we describe use of the zebrafish embryo as a model system to identify determinants of the in vivo response to ionizing radiation-induced DNA damage. To demonstrate the utility of the model we cloned and characterized the embryonic function of the XRCC5 gene, which encodes Ku80, an essential component of the nonhomologous end joining pathway of DNA repair. After the onset of zygotic transcription, Ku80 mRNA accumulates in a tissue-specific pattern, which includes proliferative zones of the retina and central nervous system. In the absence of genotoxic stress, zebrafish embryos with reduced Ku80 function develop normally. However, low dose irradiation of these embryos during gastrulation leads to marked apoptosis throughout the developing central nervous system. Apoptosis is p53 dependent, indicating that it is a downstream consequence of unrepaired DNA damage. Results suggest that nonhomologous end joining components mediate DNA repair to promote survival of irradiated cells during embryogenesis. PMID:15914672

  7. DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity.

    PubMed

    Sikdar, Nilabja; Banerjee, Soma; Lee, Kyoo-young; Wincovitch, Stephen; Pak, Evgenia; Nakanishi, Koji; Jasin, Maria; Dutra, Amalia; Myung, Kyungjae

    2009-10-01

    Genomic integrity depends on DNA replication, recombination and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of gamma-H2AX, 53BP1 and phosphorylated-ATM that mark chromosomal breaks. Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were upregulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.

  8. 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

  9. 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.

  10. Constant rate of p53 tetramerization in response to DNA damage controls the p53 response

    PubMed Central

    Gaglia, Giorgio; Lahav, Galit

    2014-01-01

    The dynamics of the tumor suppressor protein p53 have been previously investigated in single cells using fluorescently tagged p53. Such approach reports on the total abundance of p53 but does not provide a measure for functional p53. We used fluorescent protein-fragment complementation assay (PCA) to quantify in single cells the dynamics of p53 tetramers, the functional units of p53. We found that while total p53 increases proportionally to the input strength, p53 tetramers are formed in cells at a constant rate. This breaks the linear input–output relation and dampens the p53 response. Disruption of the p53-binding protein ARC led to a dose-dependent rate of tetramers formation, resulting in enhanced tetramerization and induction of p53 target genes. Our work suggests that constraining the p53 response in face of variable inputs may protect cells from committing to terminal outcomes and highlights the importance of quantifying the active form of signaling molecules in single cells. Quantification of the dynamics of p53 tetramers in single cells using a fluorescent protein-fragment complementation assay reveals that, while total p53 increases proportionally to the DNA damage strength, p53 tetramers are formed at a constant rate. PMID:25344068

  11. 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

  12. 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

  13. Tof1p regulates DNA damage responses during S phase in Saccharomyces cerevisiae.

    PubMed Central

    Foss, E J

    2001-01-01

    A tof1 mutant was recovered in a screen aimed at identifying genes involved specifically in the S phase branch of the MEC1-dependent DNA damage response pathway. The screen was based on the observation that mutants missing this branch are particularly dependent on the cell cycle-wide branch and, therefore, on RAD9, for surviving DNA damage. tof1 and rad9 conferred synergistic sensitivity to MMS, UV, and HU, and the double mutant was incapable of slowing S phase in response to MMS, inducing RNR3 transcription in response to UV, and phosphorylating Rad53p in response to HU. TOF1's contribution to DNA damage response appeared to be restricted to S phase, since TOF1 did not contribute to UV-induced transcription during G1 or to the cdc13-1-induced block to anaphase in G2/M. I suggest a model in which Tof1p functions to link Mec1p with Rad53p. PMID:11156979

  14. DNA damage in germ cells induces an innate immune response that triggers systemic stress resistance.

    PubMed

    Ermolaeva, Maria A; Segref, Alexandra; Dakhovnik, Alexander; Ou, Hui-Ling; Schneider, Jennifer I; Utermöhlen, Olaf; Hoppe, Thorsten; Schumacher, Björn

    2013-09-19

    DNA damage responses have been well characterized with regard to their cell-autonomous checkpoint functions leading to cell cycle arrest, senescence and apoptosis. In contrast, systemic responses to tissue-specific genome instability remain poorly understood. In adult Caenorhabditis elegans worms germ cells undergo mitotic and meiotic cell divisions, whereas somatic tissues are entirely post-mitotic. Consequently, DNA damage checkpoints function specifically in the germ line, whereas somatic tissues in adult C. elegans are highly radio-resistant. Some DNA repair systems such as global-genome nucleotide excision repair (GG-NER) remove lesions specifically in germ cells. Here we investigated how genome instability in germ cells affects somatic tissues in C. elegans. We show that exogenous and endogenous DNA damage in germ cells evokes elevated resistance to heat and oxidative stress. The somatic stress resistance is mediated by the ERK MAP kinase MPK-1 in germ cells that triggers the induction of putative secreted peptides associated with innate immunity. The innate immune response leads to activation of the ubiquitin-proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance. We propose that elevated systemic stress resistance promotes endurance of somatic tissues to allow delay of progeny production when germ cells are genomically compromised.

  15. Interplay Between Histone H3 Lysine 56 Deacetylation and Chromatin Modifiers in Response to DNA Damage

    PubMed Central

    Simoneau, Antoine; Delgoshaie, Neda; Celic, Ivana; Dai, Junbiao; Abshiru, Nebiyu; Costantino, Santiago; Thibault, Pierre; Boeke, Jef D.; Verreault, Alain; Wurtele, Hugo

    2015-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage–induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage–induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage. PMID:25786853

  16. 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

  17. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

    PubMed

    Reinhardt, H Christian; Yaffe, Michael B

    2013-09-01

    Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

  18. 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

    2015-01-08

    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.

  19. Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways.

    PubMed

    Ichijima, Yosuke; Sin, Ho-Su; Namekawa, Satoshi H

    2012-08-01

    Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR pathway recognizing DNA replication stress in the S phase. Additionally, it is likely to be distinct from the DDR pathway that recognizes meiosis-specific double-strand breaks. This review article extensively discusses the underlying mechanism of sex chromosome inactivation.

  20. 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.

  1. DNA damage response pathway and replication fork stress during oligonucleotide directed gene editing.

    PubMed

    Bonner, Melissa; Strouse, Bryan; Applegate, Mindy; Livingston, Paula; Kmiec, Eric B

    2012-04-03

    Single-stranded DNA oligonucleotides (ODNs) can be used to direct the exchange of nucleotides in the genome of mammalian cells in a process known as gene editing. Once refined, gene editing should become a viable option for gene therapy and molecular medicine. Gene editing is regulated by a number of DNA recombination and repair pathways whose natural activities often lead to single- and double-stranded DNA breaks. It has been previously shown that introduction of a phosphorotioated ODN, designed to direct a gene-editing event, into cells results in the activation of γH2AX, a well-recognized protein biomarker for double-stranded DNA breakage. Using a single copy, integrated mutant enhanced green fluorescent protein (eGFP) gene as our target, we now demonstrate that several types of ODNs, capable of directing gene editing, also activate the DNA damage response and the post-translational modification of proliferating cell nuclear antigen (PCNA), a signature modification of replication stress. We find that the gene editing reaction itself leads to transient DNA breakage, perhaps through replication fork collapse. Unmodified specific ODNs elicit a lesser degree of replication stress than their chemically modified counterparts, but are also less active in gene editing. Modified phosphothioate oligonucleotides (PTOs) are detrimental irrespective of the DNA sequence. Such collateral damage may prove problematic for proliferation of human cells genetically modified by gene editing.

  2. 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.

  3. Proteomics insights into DNA damage response and translating this knowledge to clinical strategies

    PubMed Central

    Olsen, Jesper V.

    2016-01-01

    Genomic instability is a critical driver in the process of cancer formation. At the same time, inducing DNA damage by irradiation or genotoxic compounds constitutes a key therapeutic strategy to kill fast‐dividing cancer cells. Sensing of DNA lesions initiates a complex set of signalling pathways, collectively known as the DNA damage response (DDR). Deciphering DDR signalling pathways with high‐throughput technologies could provide insights into oncogenic transformation, metastasis formation and therapy responses, and could build a basis for better therapeutic interventions in cancer treatment. Mass spectrometry (MS)‐based proteomics emerged as a method of choice for global studies of proteins and their posttranslational modifications (PTMs). MS‐based studies of the DDR have aided in delineating DNA damage‐induced signalling responses. Those studies identified changes in abundance, interactions and modification of proteins in the context of genotoxic stress. Here we review ground‐breaking MS‐based proteomics studies, which analysed changes in protein abundance, protein‐protein and protein‐DNA interactions, phosphorylation, acetylation, ubiquitylation, SUMOylation and Poly(ADP‐ribose)ylation (PARylation) in the DDR. Finally, we provide an outlook on how proteomics studies of the DDR could aid clinical developments on multiple levels. PMID:27682984

  4. Transcription profile of DNA damage response genes at G₀ lymphocytes exposed to gamma radiation.

    PubMed

    Saini, Divyalakshmi; Shelke, Shridevi; Mani Vannan, A; Toprani, Sneh; Jain, Vinay; Das, Birajalaxmi; Seshadri, M

    2012-05-01

    Ionizing radiation induces a plethora of DNA damages in human cells which may alter the level of mRNA expression. We have analyzed mRNA expression profile of DNA damage response genes involved in G(0)/G(1) check point pathway in whole blood to assess their radio-adaptive response, if any, to gamma radiation. Blood samples were collected from twenty-five random, normal, and healthy male donors with written informed consent and irradiated at doses between 0.1 and 2.0 Gy (0.7 Gy/min). DNA strand breaks were studied using comet assay, whereas DNA double-strand breaks were visualized using γH2AX as a biomarker. Dose response if any, at transcriptional level was studied for all these dose groups at 1 and 5-h post-irradiation. Adaptive response at transcriptional level was studied at three different priming doses (0.1, 0.3, and 0.6 Gy) separately followed by a challenging dose of 2.0 Gy after 4 h. For both the experiments, total RNA was isolated from PBMCs obtained from irradiated whole blood and reverse transcribed to cDNA. The level of mRNA expression of ATM, ATR, GADD45A, CDKN1A, P53, CDK2, MDM2, and Cyclin E was studied using real-time quantitative PCR. A significant dose-dependant increase in the percentage of DNA damage in tail was observed using comet assay. Similarly, increased number of foci was observed at γH2AX with increasing dose. At transcriptional level, a significant dose-dependent up-regulation at GADD45A, CDKN1A, and P53 genes up to 1.0 Gy was observed at 5-h post-irradiation (P ≤ 0.05). Radio-adaptive response at mRNA expression level was observed at CDK2, Cyclin E, and P53, whereas ATM, ATR, GADD45A, MDM2, ATM, and ATR have not shown any radio-adaptive changes in the expression profile. DNA damage response genes involved in G(0)/G(1) checkpoint pathway has important implications in terms of radiosensitivity in vivo and changes in the transcriptional profile might throw some new insights to understand the mechanism of adaptive response.

  5. 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

  6. 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.

  7. 14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response.

    PubMed

    Mohammad, Duaa H; Yaffe, Michael B

    2009-09-02

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phosphorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G(2)/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

  8. Modulation of the DNA damage response during the life cycle of human papillomaviruses.

    PubMed

    Anacker, Daniel C; Moody, Cary A

    2017-03-02

    Human papillomavirus (HPV) is the most common sexually transmitted viral infection. Infection with certain types of HPV pose a major public health risk as these types are associated with multiple human cancers, including cervical cancer, other anogenital malignancies and an increasing number of head and neck cancers. The HPV life cycle is closely tied to host cell differentiation with late viral events such as structural gene expression and viral genome amplification taking place in the upper layers of the stratified epithelium. The DNA damage response (DDR) is an elaborate signaling network of proteins that regulate the fidelity of replication by detecting, signaling and repairing DNA lesions. ATM and ATR are two kinases that are major regulators of DNA damage detection and repair. A multitude of studies indicate that activation of the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related) pathways are critical for HPV to productively replicate. This review outlines how HPV interfaces with the ATM- and ATR-dependent DNA damage responses throughout the viral life cycle to create an environment supportive of viral replication and how activation of these pathways could impact genomic stability.

  9. Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model.

    PubMed

    Mondesert, Odile; Frongia, Céline; Clayton, Olivia; Boizeau, Marie-Laure; Lobjois, Valérie; Ducommun, Bernard

    2015-01-01

    Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.

  10. Regulation of ATM-Dependent DNA Damage Responses in Breast Cancer by the RhoGEF Net1

    DTIC Science & Technology

    2015-05-01

    Award Number: W81XWH-12-1-0014 TITLE: Regulation of ATM-Dependent DNA Damage Responses in Breast Cancer by the RhoGEF Net1 PRINCIPAL INVESTIGATOR... DNA breaks that are lethal to a cell if not repaired. In response to IR exposure a signal transduction cascade is initiated that activates cell...cycle checkpoints to cause cell cycle arrest, thereby allowing cells time to repair their damaged DNA (3). The ability of a cell to efficiently respond

  11. UVB and caffeine: inhibiting the DNA damage response to protect against the adverse effects of UVB.

    PubMed

    Kerzendorfer, Claudia; O'Driscoll, Mark

    2009-07-01

    The incidence of sunlight-induced skin cancer is increasing. Mouse studies indicate that caffeine, administered orally or topically, promotes apoptosis of UVB-irradiated keratinocytes. In this issue, Heffernan and colleagues identify the pathway targeted by caffeine and suggest that inhibition of this DNA damage response may offer a viable therapeutic option for nonmelanoma skin cancer. This potentially represents an important protective or therapeutic option from the most unlikely of sources: your daily coffee.

  12. Topoisomerase IIalpha-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.

    PubMed

    Soubeyrand, Sébastien; Pope, Louise; Haché, Robert J G

    2010-02-01

    Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25muM etoposide was highly toxic and initiated a double-stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA-PKcs to gammaH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and gammaH2AX response to correlate with the inability of the cell to correct topoIIalpha-initiated DNA damage. gammaH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre-treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIalpha-dependent DNA defects within topologically open chromatin domains.

  13. The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast.

    PubMed Central

    Longhese, M P; Paciotti, V; Fraschini, R; Zaccarini, R; Plevani, P; Lucchini, G

    1997-01-01

    The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase. Deletion of DDC1 causes sensitivity to UV radiation, methyl methanesulfonate (MMS) and hydroxyurea (HU). ddc1Delta mutants are defective in delaying G1-S and G2-M transition and in slowing down the rate of DNA synthesis when DNA is damaged during G1, G2 or S phase, respectively. Therefore, DDC1 is involved in all the known DNA damage checkpoints. Conversely, Ddc1p is not required for delaying entry into mitosis when DNA synthesis is inhibited. ddc1 and mec3 mutants belong to the same epistasis group, and DDC1 overexpression can partially suppress MMS and HU sensitivity of mec3Delta strains, as well as their checkpoint defects. Moreover, Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage. Both phosphorylation events are at least partially dependent on a functional MEC3 gene. PMID:9311982

  14. The DNA damage response induced by infection with human cytomegalovirus and other viruses.

    PubMed

    Xiaofei, E; Kowalik, Timothy F

    2014-05-23

    Viruses use different strategies to overcome the host defense system. Recent studies have shown that viruses can induce DNA damage response (DDR). Many of these viruses use DDR signaling to benefit their replication, while other viruses block or inactivate DDR signaling. This review focuses on the effects of DDR and DNA repair on human cytomegalovirus (HCMV) replication. Here, we review the DDR induced by HCMV infection and its similarities and differences to DDR induced by other viruses. As DDR signaling pathways are critical for the replication of many viruses, blocking these pathways may represent novel therapeutic opportunities for the treatment of certain infectious diseases. Lastly, future perspectives in the field are discussed.

  15. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    PubMed

    Becherel, Olivier J; Yeo, Abrey J; Stellati, Alissa; Heng, Evelyn Y H; Luff, John; Suraweera, Amila M; Woods, Rick; Fleming, Jean; Carrie, Dianne; McKinney, Kristine; Xu, Xiaoling; Deng, Chuxia; Lavin, Martin F

    2013-04-01

    Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  16. 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

  17. Mismatch Repair Proteins Are Activators of Toxic Responses to Chromium-DNA Damage

    PubMed Central

    Peterson-Roth, Elizabeth; Reynolds, Mindy; Quievryn, George; Zhitkovich, Anatoly

    2005-01-01

    Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of γ-H2AX foci in G2 phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of γ-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G2 arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers. PMID:15831465

  18. DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

    PubMed

    Dutertre, Martin; Vagner, Stéphan

    2016-09-29

    Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs.

  19. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication.

    PubMed

    Mitchell, Jonathan K; Friesen, Paul D

    2012-12-01

    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.

  20. Ubiquitin-binding domains and their role in the DNA damage response.

    PubMed

    Hofmann, Kay

    2009-04-05

    The modification of eukaryotic proteins by covalent attachment of ubiquitin is a versatile signaling event with a wide range of possible consequences. Canonical poly-ubiquitination by Lys-48 linked chains usually destines a protein for degradation by the proteasome. By contrast, attachment of a single ubiquitin or ubiquitin chains linked through Lys-63 or Lys-6 serves a non-proteolytic role. Over the last years, evidence has accumulated that several nuclear proteins become ubiquitinated in response to DNA damage. Typically, these proteins carry mono-ubiquitin or non-classical ubiquitin chains and are localized close to the site of DNA damage. Of particular interest are PCNA and the variant histone H2AX, two key proteins whose ubiquitination serves to recruit factors needed by the cell to cope with the damage. A prerequisite for docking effector proteins to the site of the lesion is the detection of a specific ubiquitin modification, a process that can be mediated by a range of dedicated ubiquitin-binding domains (UBDs). As the same types of ubiquitin modification are involved in entirely different processes, the recognition of the ubiquitin mark has to go along with the recognition of the modified protein. Thus, ubiquitin-binding domains gain their specificity through combination with other recognition domains and motifs. This review discusses ubiquitin-binding domains relevant to the DNA damage response, including their binding mode, their specificity, and their interdependence with other factors. For several repair pathways, current knowledge of the events downstream of the ubiquitin mark is sketchy. A closer look at orphan UBD proteins might lead to the identification of missing pieces in the DNA response puzzle.

  1. 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

  2. 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

  3. 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

  4. G2E3 IS A NUCLEO-CYTOPLASMIC SHUTTLING PROTEIN WITH DNA DAMAGE RESPONSIVE LOCALIZATION

    PubMed Central

    Brooks, William S.; Banerjee, Sami; Crawford, David F.

    2007-01-01

    G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as an ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage. PMID:17239372

  5. G2E3 is a nucleo-cytoplasmic shuttling protein with DNA damage responsive localization

    SciTech Connect

    Brooks, William S.; Banerjee, Sami; Crawford, David F. . E-mail: dfc@uab.edu

    2007-02-15

    G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as a ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage.

  6. 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

  7. Carbon nanotubes elicit DNA damage and inflammatory response relative to their size and shape.

    PubMed

    Yamashita, Kohei; Yoshioka, Yasuo; Higashisaka, Kazuma; Morishita, Yuki; Yoshida, Tokuyuki; Fujimura, Maho; Kayamuro, Hiroyuki; Nabeshi, Hiromi; Yamashita, Takuya; Nagano, Kazuya; Abe, Yasuhiro; Kamada, Haruhiko; Kawai, Yuichi; Mayumi, Tadanori; Yoshikawa, Tomoaki; Itoh, Norio; Tsunoda, Shin-ichi; Tsutsumi, Yasuo

    2010-08-01

    Carbon nanotubes (CNTs) have been one of the most extensively researched and developed nanomaterials. However, little concern has been placed on their safety. The biological effects of CNTs are believed to differ relative to size and shape. Thus, the relationship between the characteristics of CNTs and their safety needs to be evaluated. In this study, we examined the biological effects of different-sized multi-walled CNTs (MWCNTs) and single-walled CNTs (SWCNTs). Long and thick MWCNTs induced the strongest DNA damage while similar SWCNTs caused little effect. Comparison of inflammatory responses of various types of CNTs found that peritoneal CNT administration of long and thick MWCNTs increased the total cell number in abdominal lavage fluid in mice. These results indicate that long and thick MWCNT, but not short and thin MWCNT, cause DNA damage and severe inflammatory effects. These findings might provide useful information for constructing novel CNTs with safety.

  8. The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.

    PubMed

    Puente, Bao N; Kimura, Wataru; Muralidhar, Shalini A; Moon, Jesung; Amatruda, James F; Phelps, Kate L; Grinsfelder, David; Rothermel, Beverly A; Chen, Rui; Garcia, Joseph A; Santos, Celio X; Thet, SuWannee; Mori, Eiichiro; Kinter, Michael T; Rindler, Paul M; Zacchigna, Serena; Mukherjee, Shibani; Chen, David J; Mahmoud, Ahmed I; Giacca, Mauro; Rabinovitch, Peter S; Aroumougame, Asaithamby; Shah, Ajay M; Szweda, Luke I; Sadek, Hesham A

    2014-04-24

    The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

  9. 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

  10. Mitochondrial DNA damage and a hypoxic response are induced by CoCl2 in rat neuronal PC12 cells

    PubMed Central

    Wang, Guichun; Hazra, Tapas K.; Mitra, Sankar; Lee, Heung-Man; Englander, Ella W.

    2000-01-01

    Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl2, mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 µM CoCl2. In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1α (HIF-1α), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage. PMID:10773083

  11. Mitochondrial DNA damage and a hypoxic response are induced by CoCl(2) in rat neuronal PC12 cells.

    PubMed

    Wang, G; Hazra, T K; Mitra, S; Lee, H M; Englander, E W

    2000-05-15

    Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl(2), mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 microM CoCl(2). In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1alpha(HIF-1alpha), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage.

  12. Aberrant expression of DNA damage response proteins is associated with breast cancer subtype and clinical features

    PubMed Central

    Guler, Gulnur; Himmetoglu, Cigdem; Jimenez, Rafael E.; Geyer, Susan M.; Wang, Wenle P.; Costinean, Stefan; Pilarski, Robert T.; Morrison, Carl; Suren, Dinc; Liu, Jianhua; Chen, Jingchun; Kamal, Jyoti; Shapiro, Charles L.

    2013-01-01

    Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins γH2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2α, Ap2γ, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2γ protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread. PMID:21069451

  13. Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

    PubMed

    Huelsenbeck, Stefanie C; Schorr, Anne; Roos, Wynand P; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

    2012-11-09

    To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

  14. 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.

  15. DNA damage response regulation by microRNAs as a therapeutic target in cancer.

    PubMed

    Majidinia, Maryam; Yousefi, Bahman

    2016-11-01

    The inability of cancer cells in taking care of DNA damages can lead to cancer development and/or progression. Due to the essential role of DNA repair in maintaining genomic stability, tightly controlled regulatory mechanism are required for these processes. Recent studies have shown a myriad of interactions among DNA damage response (DDR) components and miRNAs. While DDR modulates miRNA expression in transcriptional and post-transcriptional levels and affects miRNA degradation, miRNAs in turn, directly modulate the expression of multiple proteins in the DDR pathways, or indirectly fine-tune the expression of such proteins. A better understanding of DDR-miRNA interactions can facilitate the development of new anticancer agents targeting miRNAs involved in the DNA repair process. In this review, we provide a brief introduction about miRNA biogenesis and functions, DDR pathways, and recent findings about DDR-microRNA interactions. Finally, the therapeutic importance of miRNAs in modulation of DDR/DNA repair mechanisms will be discussed.

  16. Polo-like kinase 1 inhibits DNA damage response during mitosis

    PubMed Central

    Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

    2015-01-01

    In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis. PMID:25607646

  17. Polo-like kinase 1 inhibits DNA damage response during mitosis.

    PubMed

    Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

    2015-01-01

    In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

  18. NAD homeostasis in the bacterial response to DNA/RNA damage.

    PubMed

    Sorci, Leonardo; Ruggieri, Silverio; Raffaelli, Nadia

    2014-11-01

    In mammals, NAD represents a nodal point for metabolic regulation, and its availability is critical to genome stability. Several NAD-consuming enzymes are induced in various stress conditions and the consequent NAD decline is generally accompanied by the activation of NAD biosynthetic pathways to guarantee NAD homeostasis. In the bacterial world a similar scenario has only recently begun to surface. Here we review the current knowledge on the involvement of NAD homeostasis in bacterial stress response mechanisms. In particular, we focus on the participation of both NAD-consuming enzymes (DNA ligase, mono(ADP-ribosyl) transferase, sirtuins, and RNA 2'-phosphotransferase) and NAD biosynthetic enzymes (both de novo, and recycling enzymes) in the response to DNA/RNA damage. As further supporting evidence for such a link, a genomic context analysis is presented showing several conserved associations between NAD homeostasis and stress responsive genes.

  19. Genetic Interaction Landscape Reveals Critical Requirements for Schizosaccharomyces pombe Brc1 in DNA Damage Response Mutants

    PubMed Central

    Sánchez, Arancha; Roguev, Assen; Krogan, Nevan J.; Russell, Paul

    2015-01-01

    Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN). Exogenous genotoxins enhance these negative genetic interactions. Rad52 and RPA foci are increased in CSN-defective cells, and loss of γH2A increases genotoxin sensitivity, indicating a critical role for the γH2A-Brc1 module in stabilizing replication forks in CSN-defective cells. A negative genetic interaction with the Nse6 subunit of Smc5-Smc6 holocomplex indicates that the DNA repair functions of Brc1 and Smc5-Smc6 holocomplex are at least partially independent. Rtt107, the Brc1 homolog in Saccharomyces cerevisiae, has a very different pattern of genetic interactions, indicating evolutionary divergence of functions and DNA damage responses. PMID:25795664

  20. 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

  1. Vaccinia-related kinase 1 (VRK1) confers resistance to DNA-damaging agents in human breast cancer by affecting DNA damage response

    PubMed Central

    Salzano, Marcella; Vázquez-Cedeira, Marta; Sanz-García, Marta; Valbuena, Alberto; Blanco, Sandra; Fernández, Isabel F.; Lazo, Pedro A.

    2014-01-01

    Vaccinia-related kinase 1 (VRK1) belongs to a group of sixteen kinases associated to a poorer prognosis in human breast carcinomas, particularly in estrogen receptor positive cases based on gene expression arrays. In this work we have studied the potential molecular mechanism by which the VRK1 protein can contribute to a poorer prognosis in this disease. For this aim it was first analyzed by immunohistochemistry the VRK1 protein level in normal breast and in one hundred and thirty six cases of human breast cancer. The effect of VRK1 to protect against DNA damage was determined by studying the effect of its knockdown on the formation of DNA repair foci assembled on 53BP1 in response to treatment with ionizing radiation or doxorubicin in two breast cancer cell lines. VRK1 protein was detected in normal breast and in breast carcinomas at high levels in ER and PR positive tumors. VRK1 protein level was significantly lower in ERBB2 positive cases. Next, to identify a mechanism that can link VRK1 to poorer prognosis, VRK1 was knocked-down in two breast cancer cell lines that were treated with ionizing radiation or doxorubicin, both inducing DNA damage. Loss of VRK1 resulted in reduced formation of DNA-damage repair foci complexes assembled on the 53BP1 scaffold protein, and this effect was independent of damaging agent or cell type. This observation is consistent with detection of high VRK1 protein levels in ER and PR positive breast cancers. We conclude that VRK1 can contribute to make these tumors more resistant to DNA damage-based therapies, such as ionizing radiation or doxorubicin, which is consistent with its association to a poor prognosis in ER positive breast cancer. VRK1 is potential target kinase for development of new specific inhibitors which can facilitate sensitization to other treatments in combination therapies; or alternatively be used as a new cancer drugs. PMID:24731990

  2. The interplay among chromatin dynamics, cell cycle checkpoints and repair mechanisms modulates the cellular response to DNA damage.

    PubMed

    Lazzaro, Federico; Giannattasio, Michele; Muzi-Falconi, Marco; Plevani, Paolo

    2007-06-01

    Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.

  3. 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

  4. SIRT4 has tumor suppressive activity and regulates the cellular metabolic response to DNA damage by inhibiting mitochondrial glutamine metabolism

    PubMed Central

    Jeong, Seung Min; Xiao, Cuiying; Finley, Lydia W.S; Lahusen, Tyler; Souza, Amanda L.; Pierce, Kerry; Li, Ying-Hua; Wang, Xiaoxu; Laurent, Gaëlle; German, Natalie J.; Xu, Xiaoling; Li, Cuiling; Wang, Rui-Hong; Lee, Jaewon; Csibi, Alfredo; Cerione, Richard; Blenis, John; Clish, Clary B.; Kimmelman, Alec; Deng, Chu-Xia; Haigis, Marcia C.

    2013-01-01

    SUMMARY DNA damage elicits a cellular signaling response that initiates cell cycle arrest and DNA repair. Here we find that DNA damage triggers a critical block in glutamine metabolism, which is required for proper DNA damage responses. This block requires the mitochondrial SIRT4, which is induced by numerous genotoxic agents and represses the metabolism of glutamine into TCA cycle. SIRT4 loss leads to both increased glutamine-dependent proliferation and stress-induced genomic instability, resulting in tumorigenic phenotypes. Moreover, SIRT4 knockout mice spontaneously develop lung tumors. Our data uncover SIRT4 as an important component of the DNA damage response pathway that orchestrates a metabolic block in glutamine metabolism, cell cycle arrest and tumor suppression. PMID:23562301

  5. DNA Damage Response Assessments in Human Tumor Samples Provide Functional Biomarkers of Radiosensitivity

    PubMed Central

    Willers, Henning; Gheorghiu, Liliana; Liu, Qi; Efstathiou, Jason A.; Wirth, Lori J.; Krause, Mechthild; von Neubeck, Cläre

    2015-01-01

    Predictive biomarkers are urgently needed for individualization of radiation therapy and treatment with radiosensitizing anti-cancer agents. Genomic profiling of human cancers will provide us with unprecedented insight into the mutational landscape of genes directly or indirectly involved in the response to radiation-induced DNA damage. However, to what extent this wealth of structural information about the cancer genome will produce biomarkers of sensitivity to radiation remains to be seen. Investigators are increasingly studying the subnuclear accumulation (i.e., foci) of proteins in the DNA damage response (DDR), such as γ-H2AX, 53BP1, or RAD51, as a surrogate of treatment sensitivity. Recent findings from preclinical studies have demonstrated the predictive potential of DDR foci by correlating foci with clinically relevant endpoints such as tumor control probability. Therefore, pre-clinical investigations of DDR foci responses are increasingly moving into cells and tissues from patients, which is the major focus of this review. The advantage of using DDR foci as functional biomarkers is that they can detect alterations in DNA repair due to various mechanisms. Moreover, they provide a global measurement of DDR network function without needing to know the identities of all the components, many of which remain unknown. Foci assays are thus expected to yield functional insight that may complement or supersede genomic information, thereby giving radiation oncologists unique opportunities to individualize cancer treatments in the near future. PMID:26384272

  6. Stop pulling my strings — what telomeres taught us about the DNA damage response

    PubMed Central

    Lazzerini-Denchi, Eros; Sfeir, Agnel

    2017-01-01

    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

  7. Replicating damaged DNA in eukaryotes.

    PubMed

    Chatterjee, Nimrat; Siede, Wolfram

    2013-12-01

    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.

  8. 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.

  9. Recurrent Initiation: A Mechanism for Triggering p53 Pulses in Response to DNA Damage

    PubMed Central

    Batchelor, Eric; Mock, Caroline; Bhan, Irun; Loewer, Alexander; Lahav, Galit

    2008-01-01

    SUMMARY DNA damage initiates a series of p53 pulses. Although much is known about the interactions surrounding p53, little is known about which interactions contribute to p53’s dynamical behavior. The simplest explanation is that these pulses are oscillations intrinsic to the p53/Mdm2 negative feedback loop. Here we present evidence that this simple mechanism is insufficient to explain p53 pulses; we show that p53 pulses are externally driven by pulses in the upstream signaling kinases, ATM and Chk2, and that the negative feedback between p53 and ATM, via Wip1, is essential for maintaining the uniform shape of p53 pulses. We propose that p53 pulses result from repeated initiation by ATM which is re-activated by persistent DNA damage. Our study emphasizes the importance of collecting quantitative dynamic information at high temporal resolution for understanding the regulation of signaling pathways and opens new ways to manipulate p53 pulses to ask questions about their function in response to DNA damage. PMID:18471974

  10. Exposure to copper induces oxidative and stress responses and DNA damage in the coral Montastraea franksi.

    PubMed

    Schwarz, J A; Mitchelmore, C L; Jones, R; O'Dea, A; Seymour, S

    2013-04-01

    Copper is a common chemical contaminant in coastal environments, including coral reefs. Ecotoxicological studies have demonstrated that exposure to copper can cause stress and detrimental effects in both host cnidarian and algal symbionts. The objective of this study was to investigate the sublethal effects of copper on the reef-building coral Montastraea franksi, by identifying genes with altered expression in corals exposed to dissolved copper, and by measuring the extent of damage to DNA in response to copper exposure. Corals exposed to 30 μg L(-1) copper for 48 h experienced significant DNA damage and displayed changes in expression patterns of genes that are known to play role cellular and oxidative stress responses. Corals also experienced changes in gene expression of genes that are not already known to play roles in oxidative stress in corals. Our data suggest that these genes may either play roles directly in mediating a stress response, or may be genes acting downstream of the stress response. These include an ETS domain-containing transcription factor related to the ETS1 family of transcription factors, known in mammals to mediate development, disease, and stress response, and two genes that are associated with biomineralization: galaxin, a protein from the organic matrix of the coral skeleton, and a coral-specific gene SCRIP2.

  11. Host DNA Damage Response Factors Localize to Merkel Cell Polyomavirus DNA Replication Sites To Support Efficient Viral DNA Replication

    PubMed Central

    Tsang, Sabrina H.; Wang, Xin; Li, Jing; Buck, Christopher B.

    2014-01-01

    ABSTRACT Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle. IMPORTANCE MCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM- and ATR

  12. 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

  13. EdU induces DNA damage response and cell death in mESC in culture.

    PubMed

    Kohlmeier, Fanni; Maya-Mendoza, Apolinar; Jackson, Dean A

    2013-03-01

    Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

  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. Site-specific DICER and DROSHA RNA products control the DNA-damage response.

    PubMed

    Francia, Sofia; Michelini, Flavia; Saxena, Alka; Tang, Dave; de Hoon, Michiel; Anelli, Viviana; Mione, Marina; Carninci, Piero; d'Adda di Fagagna, Fabrizio

    2012-08-09

    Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11–RAD50–NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.

  16. RAD50 targeting impairs DNA damage response and sensitizes human breast cancer cells to cisplatin therapy

    PubMed Central

    Flores-Pérez, Ali; Rafaelli, Lourdes E; Ramírez-Torres, Nayeli; Aréchaga-Ocampo, Elena; Frías, Sara; Sánchez, Silvia; Marchat, Laurence A; Hidalgo-Miranda, Alfredo; Quintanar-Jurado, Valeria; Rodríguez-Cuevas, Sergio; Bautista-Piña, Verónica; Carlos-Reyes, Ángeles; López-Camarillo, César

    2014-01-01

    In tumor cells the effectiveness of anti-neoplastic agents that cause cell death by induction of DNA damage is influenced by DNA repair activity. RAD50 protein plays key roles in DNA double strand breaks repair (DSBs), which is crucial to safeguard genome integrity and sustain tumor suppression. However, its role as a potential therapeutic target has not been addressed in breast cancer. Our aim in the present study was to analyze the expression of RAD50 protein in breast tumors, and evaluate the effects of RAD50-targeted inhibition on the cytotoxicity exerted by cisplatin and anthracycline and taxane-based therapies in breast cancer cells. Immunohistochemistry assays on tissue microarrays indicate that the strong staining intensity of RAD50 was reduced in 14% of breast carcinomas in comparison with normal tissues. Remarkably, RAD50 silencing by RNA interference significantly enhanced the cytotoxicity of cisplatin. Combinations of cisplatin with doxorubicin and paclitaxel drugs induced synergistic effects in early cell death of RAD50-deficient MCF-7, SKBR3, and T47D breast cancer cells. Furthermore, we found an increase in the number of DSBs, and delayed phosphorylation of histone H2AX after cisplatin treatment in RAD50-silenced cells. These cellular events were associated to a dramatical increase in the frequency of chromosomal aberrations and a decrease of cell number in metaphase. In conclusion, our data showed that RAD50 abrogation impairs DNA damage response and sensitizes breast cancer cells to cisplatin-combined therapies. We propose that the development and use of inhibitors to manipulate RAD50 levels might represent a promising strategy to sensitize breast cancer cells to DNA damaging agents. PMID:24642965

  17. Probing the Dynamic Interaction between Damaged DNA and a Cellular Responsive Protein Using a Piezoelectric Mass Biosensor.

    PubMed

    Jin, Yulong; Xie, Yunfeng; Wu, Kui; Huang, Yanyan; Wang, Fuyi; Zhao, Rui

    2017-03-15

    The binding events between damaged DNA and recognition biomolecules are of great interest for understanding the activity of DNA-damaging drugs and the related DNA repair networks. Herein, a simple and sensitive sensor system was tailored for real-time probing of the dynamic molecular recognition between cisplatin-damaged-DNA (cisPt-DNA) and a cellular responsive protein, high-mobility-group box 1 (HMGB1). By integration of flow injection analysis (FIA) with quartz crystal microbalance (QCM), the interaction time-course of cisPt-DNA and HMGB1 domain A (HMGB1a) was investigated. The highly specific sensing interface was carefully designed and fabricated using cisPt-DNA as recognition element. A hybrid self-assembled monolayer consisting of cysteamine and mercaptohexanol was introduced to resist nonspecific adsorption. The calculated kinetic parameters (kass and kdiss) and the dissociation constant (KD) demonstrated the rapid recognition and tight binding of HMGB1a toward cisPt-DNA. Molecular docking was employed to simulate the complex formed by cisPt-DNA and HMGB1a. The tight binding of such a DNA-damage responsive complex is appealing for the downstream molecular recognition event related to the resistance to DNA repair. This continuous-flow QCM biosensor is an ideal tool for studying specific interactions between drug-damaged-DNAs and their recognition proteins in a physiological-relevant environment, and will provide a potential sensor platform for rapid screening and evaluating metal anticancer drugs.

  18. Real-time fluorescence imaging of the DNA damage repair response during mitosis.

    PubMed

    Miwa, Shinji; Yano, Shuya; Yamamoto, Mako; Matsumoto, Yasunori; Uehara, Fuminari; Hiroshima, Yukihiko; Toneri, Makoto; Murakami, Takashi; Kimura, Hiroaki; Hayashi, Katsuhiro; Yamamoto, Norio; Efimova, Elena V; Tsuchiya, Hiroyuki; Hoffman, Robert M

    2015-04-01

    The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe.

  19. 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.

  20. PTEN Activation by DNA Damage Induces Protective Autophagy in Response to Cucurbitacin B in Hepatocellular Carcinoma Cells

    PubMed Central

    Niu, Yanan; Sun, Wen; Lu, Jin-Jian; Pei, Lixia

    2016-01-01

    Cucurbitacin B (Cuc B), a natural product, induced both protective autophagy and DNA damage mediated by ROS while the detailed mechanisms remain unclear. This study explored the mechanism of Cuc B-induced DNA damage and autophagy. Cuc B decreased cell viability in concentration- and time-dependent manners. Cuc B caused long comet tails and increased expression of γ-H2AX, phosphorylation of ATM/ATR, and Chk1/Chk2. Cuc B induced autophagy as evidenced by monodansylcadaverine (MDC) staining, increased expression of LC3II, phosphorylated ULK1, and decreased expression of phosphorylated AKT, mTOR. Cuc B induced apoptosis mediated by Bcl-2 family proteins and caspase activation. Furthermore, Cuc B induced ROS formation, which was inhibited by N-acetyl-L-cysteine (NAC). NAC pretreatment dramatically reversed Cuc B-induced DNA damage, autophagy, and apoptosis. Cuc B-induced apoptosis was reversed by NAC but enhanced by 3-methyladenine (3-MA), chloroquine (CQ), and silencing phosphatase and tensin homolog (PTEN). 3-MA and CQ showed no effect on Cuc B-induced DNA damage. In addition, Cuc B increased PTEN phosphorylation and silence PTEN restored Cuc B-induced autophagic protein expressions without affecting DNA damage. In summary, Cuc B induced DNA damage, apoptosis, and protective autophagy mediated by ROS. PTEN activation in response to DNA damage bridged DNA damage and prosurvival autophagy. PMID:28042385

  1. 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

  2. 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.

  3. Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells.

    PubMed

    Modi, Prexy; Balakrishnan, Kumudha; Yang, Qingshan; Wierda, William G; Keating, Michael J; Gandhi, Varsha

    2017-02-07

    Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL.

  4. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response.

    PubMed

    Bennetzen, Martin V; Larsen, Dorthe Helena; Dinant, Christoffel; Watanabe, Sugiko; Bartek, Jiri; Lukas, Jiri; Andersen, Jens S

    2013-06-01

    Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.

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

    PubMed

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

    2012-09-01

    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.

  6. 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

  7. Fhit loss in lung preneoplasia: relation to DNA damage response checkpoint activation

    PubMed Central

    Cirombella, Roberto; Montrone, Giuseppe; Stoppacciaro, Antonella; Giglio, Simona; Volinia, Stefano; Graziano, Paolo; Huebner, Kay; Vecchione, Andrea

    2009-01-01

    Loss of heterozygosity at the FHIT locus is coincident with activation of DNA damage response checkpoint proteins; thus damage at fragile loci may trigger checkpoint activation. We examined preneoplastic lesions adjacent to non-small cell lung carcinomas for alterations to expression of Fhit and activated checkpoint proteins. Expression scores were analyzed for pair-wise associations and correlations among proteins and type of lesion. Hyperplastic and dysplastic lesions were positive for nuclear H2AX expression; 12/20 dysplastic lesions were negative for Fhit expression. Fhit positive lesions showed expression of most checkpoint proteins examined, while Fhit negative lesions showed absence of expression of Chk1 and phosphoChk1. The results show that loss of expression of Fhit is significantly directly correlated with absence of activated Chk1 in dysplasia, and suggest a connection between loss of Fhit and modulation of checkpoint activity. PMID:19931269

  8. Low intensity microwave radiation induced oxidative stress, inflammatory response and DNA damage in rat brain.

    PubMed

    Megha, Kanu; Deshmukh, Pravin Suryakantrao; Banerjee, Basu Dev; Tripathi, Ashok Kumar; Ahmed, Rafat; Abegaonkar, Mahesh Pandurang

    2015-12-01

    Over the past decade people have been constantly exposed to microwave radiation mainly from wireless communication devices used in day to day life. Therefore, the concerns over potential adverse effects of microwave radiation on human health are increasing. Until now no study has been proposed to investigate the underlying causes of genotoxic effects induced by low intensity microwave exposure. Thus, the present study was undertaken to determine the influence of low intensity microwave radiation on oxidative stress, inflammatory response and DNA damage in rat brain. The study was carried out on 24 male Fischer 344 rats, randomly divided into four groups (n=6 in each group): group I consisted of sham exposed (control) rats, group II-IV consisted of rats exposed to microwave radiation at frequencies 900, 1800 and 2450 MHz, specific absorption rates (SARs) 0.59, 0.58 and 0.66 mW/kg, respectively in gigahertz transverse electromagnetic (GTEM) cell for 60 days (2h/day, 5 days/week). Rats were sacrificed and decapitated to isolate hippocampus at the end of the exposure duration. Low intensity microwave exposure resulted in a frequency dependent significant increase in oxidative stress markers viz. malondialdehyde (MDA), protein carbonyl (PCO) and catalase (CAT) in microwave exposed groups in comparison to sham exposed group (p<0.05). Whereas, levels of reduced glutathione (GSH) and superoxide dismutase (SOD) were found significantly decreased in microwave exposed groups (p<0.05). A significant increase in levels of pro-inflammatory cytokines (IL-2, IL-6, TNF-α, and IFN-γ) was observed in microwave exposed animal (p<0.05). Furthermore, significant DNA damage was also observed in microwave exposed groups as compared to their corresponding values in sham exposed group (p<0.05). In conclusion, the present study suggests that low intensity microwave radiation induces oxidative stress, inflammatory response and DNA damage in brain by exerting a frequency dependent effect

  9. Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses.

    PubMed

    Kobayashi, Junya; Saito, Yuichiro; Okui, Michiyo; Miwa, Noriko; Komatsu, Kenshi

    2015-04-01

    Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration.

  10. PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks.

    PubMed

    Krietsch, Jana; Caron, Marie-Christine; Gagné, Jean-Philippe; Ethier, Chantal; Vignard, Julien; Vincent, Michel; Rouleau, Michèle; Hendzel, Michael J; Poirier, Guy G; Masson, Jean-Yves

    2012-11-01

    After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.

  11. Cell synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Halicka, H. Dorota; Zhao, Hong

    2010-01-01

    Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or thymidine become arrested at the entrance to S-phase and upon release from the block they synchronously progress through S, G2 and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response cell signaling pathways and caution about interpreting data obtained with the use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific Ab and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase. PMID:21755443

  12. Post-translational modifications of proliferating cell nuclear antigen: A key signal integrator for DNA damage response (Review).

    PubMed

    Zhu, Qiong; Chang, Yuxiao; Yang, Jin; Wei, Quanfang

    2014-05-01

    Previous studies have shown that the post-translational modifications of proliferating cell nuclear antigen (PCNA) may be crucial in influencing the cellular choice between different pathways, such as the cell cycle checkpoint, DNA repair or apoptosis pathways, in order to maintain genomic stability. DNA damage leads to replication stress and the subsequent induction of PCNA modification by small ubiquitin (Ub)-related modifiers and Ub, which has been identified to affect multiple biological processes of genomic DNA. Thus far, much has been learned concerning the behavior of modified PCNA as a key signal integrator in response to DNA damage. In humans and yeast, modified PCNA activates DNA damage bypass via an error-prone or error-free pathway to prevent the breakage of DNA replication forks, which may potentially induce double-strand breaks and subsequent chromosomal rearrangements. However, the exact mechanisms by which these pathways work and by what means the modified PCNA is involved in these processes remain elusive. Thus, the improved understanding of PCNA modification and its implications for DNA damage response may provide us with more insight into the mechanisms by which human cells regulate aberrant recombination events, and cancer initiation and development. The present review focuses on the post-translational modifications of PCNA and its important functions in mediating mammalian cellular response to different types of DNA damage.

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

    PubMed

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

    2016-01-12

    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.

  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. 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

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

    PubMed

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

    2015-08-12

    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.

  17. Bmi1 regulates mitochondrial function and the DNA damage response pathway.

    PubMed

    Liu, Jie; Cao, Liu; Chen, Jichun; Song, Shiwei; Lee, In Hye; Quijano, Celia; Liu, Hongjun; Keyvanfar, Keyvan; Chen, Haoqian; Cao, Long-Yue; Ahn, Bong-Hyun; Kumar, Neil G; Rovira, Ilsa I; Xu, Xiao-Ling; van Lohuizen, Maarten; Motoyama, Noboru; Deng, Chu-Xia; Finkel, Toren

    2009-05-21

    Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1(-/-) phenotype. Here we demonstrate that cells derived from Bmi1(-/-) mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1(-/-) mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

  18. The 3′–5′ DNA Exonuclease TREX1 Directly Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) during the DNA Damage Response*

    PubMed Central

    Miyazaki, Takuya; Kim, Yong-Soo; Yoon, Jeongheon; Wang, Hongsheng; Suzuki, Teruhiko; Morse, Herbert C.

    2014-01-01

    The main function of the 3′–5′ DNA exonuclease TREX1 is to digest cytosolic single-stranded DNA to prevent activation of cell-intrinsic responses to immunostimulatory DNA. TREX1 translocates to the nucleus following DNA damage with its nuclear activities being less well defined. Although mutations in human TREX1 have been linked to autoimmune/inflammatory diseases, the mechanisms contributing to the pathogenesis of these diseases remain incompletely understood. Here, using mass spectrometry and co-immunoprecipitation assays and in vivo overexpression models, we show that TREX1 interacts with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear enzyme involved in the DNA damage response. Two zinc finger domains at the amino terminus of PARP1 were required for the interaction with TREX1 that occurs after nuclear translocation of TREX1 in response to DNA damage. Functional studies suggested that TREX1 may contribute to stabilization of PARP1 levels in the DNA damage response and its activity. These results provide new insights into the mechanisms of single-stranded DNA repair following DNA damage and alterations induced by gene mutations. PMID:25278026

  19. Chromatin-associated proteins HMGB1/2 and PDIA3 trigger cellular response to chemotherapy-induced DNA damage

    PubMed Central

    Krynetskaia, Natalia F.; Phadke, Manali S.; Jadhav, Sachin H.; Krynetskiy, Evgeny Y

    2009-01-01

    Identification of the new molecular components of the DNA damage signaling cascade opens novel avenues to enhance efficacy of chemotherapeutic drugs. High mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to incorporation of non-natural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; heat shock 70kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked down cancer cells after 24-72 hr incubation with 1 μM cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress; and PDIA3 has been found essential for H2AX phosphorylation (no γ-H2AX was accumulated after 24-72 hr incubation with 1 μM cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53, and phosphorylation of H2AX, occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention. PMID:19372559

  20. The Role of the DNA Damage Response Kinase Ataxia Telangiectasia Mutated in Neuroprotection

    PubMed Central

    Marinoglou, Konstantina

    2012-01-01

    It has been estimated that a human cell is confronted with 1 million DNA lesions every day, one fifth of which may originate from the activity of Reactive Oxygen Species (ROS) alone [1,2]. Terminally differentiated neurons are highly active cells with, if any, very restricted regeneration potential [3]. In addition, genome integrity and maintenance during neuronal development is crucial for the organism. Therefore, highly accurate and robust mechanisms for DNA repair are vital for neuronal cells. This requirement is emphasized by the long list of human diseases with neurodegenerative phenotypes, which are either caused by or associated with impaired function of proteins involved in the cellular response to genotoxic stress [4-8]. Ataxia Telangiectasia Mutated (ATM), one of the major kinases of the DNA Damage Response (DDR), is a node that links DDR, neuronal development, and neurodegeneration [2,9-12]. In humans, inactivating mutations of ATM lead to Ataxia-Telangiectasia (A-T) disease [11,13], which is characterized by severe cerebellar neurodegeneration, indicating an important protective function of ATM in the nervous system [14]. Despite the large number of studies on the molecular cause of A-T, the neuroprotective role of ATM is not well established and is contradictory to its general proapoptotic function. This review discusses the putative functions of ATM in neuronal cells and how they might contribute to neuroprotection. PMID:23239948

  1. 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

  2. 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.

  3. 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

  4. Intracellular accumulation of indium ions released from nanoparticles induces oxidative stress, proinflammatory response and DNA damage.

    PubMed

    Tabei, Yosuke; Sonoda, Akinari; Nakajima, Yoshihiro; Biju, Vasudevanpillai; Makita, Yoji; Yoshida, Yasukazu; Horie, Masanori

    2016-02-01

    Due to the widespread use of indium tin oxide (ITO), it is important to investigate its effect on human health. In this study, we evaluated the cellular effects of ITO nanoparticles (NPs), indium chloride (InCl3) and tin chloride (SnCl3) using human lung epithelial A549 cells. Transmission electron microscopy and inductively coupled plasma mass spectrometry were employed to study cellular ITO NP uptake. Interestingly, greater uptake of ITO NPs was observed, as compared with soluble salts. ITO NP species released could be divided into two types: 'indium release ITO' or 'tin release ITO'. We incubated A549 cells with indium release ITO, tin release ITO, InCl3 or SnCl2 and investigated oxidative stress, proinflammatory response, cytotoxicity and DNA damage. We found that intracellular reactive oxygen species were increased in cells incubated with indium release ITO, but not tin release ITO, InCl3 or SnCl2. Messenger RNA and protein levels of the inflammatory marker, interleukin-8, also increased following exposure to indium release ITO. Furthermore, the alkaline comet assay revealed that intracellular accumulation of indium ions induced DNA damage. Our results demonstrate that the accumulation of ionic indium, but not ionic tin, from ITO NPs in the intracellular matrix has extensive cellular effects.

  5. Intracellular accumulation of indium ions released from nanoparticles induces oxidative stress, proinflammatory response and DNA damage

    PubMed Central

    Tabei, Yosuke; Sonoda, Akinari; Nakajima, Yoshihiro; Biju, Vasudevanpillai; Makita, Yoji; Yoshida, Yasukazu; Horie, Masanori

    2016-01-01

    Due to the widespread use of indium tin oxide (ITO), it is important to investigate its effect on human health. In this study, we evaluated the cellular effects of ITO nanoparticles (NPs), indium chloride (InCl3) and tin chloride (SnCl3) using human lung epithelial A549 cells. Transmission electron microscopy and inductively coupled plasma mass spectrometry were employed to study cellular ITO NP uptake. Interestingly, greater uptake of ITO NPs was observed, as compared with soluble salts. ITO NP species released could be divided into two types: ‘indium release ITO’ or ‘tin release ITO’. We incubated A549 cells with indium release ITO, tin release ITO, InCl3 or SnCl2 and investigated oxidative stress, proinflammatory response, cytotoxicity and DNA damage. We found that intracellular reactive oxygen species were increased in cells incubated with indium release ITO, but not tin release ITO, InCl3 or SnCl2. Messenger RNA and protein levels of the inflammatory marker, interleukin-8, also increased following exposure to indium release ITO. Furthermore, the alkaline comet assay revealed that intracellular accumulation of indium ions induced DNA damage. Our results demonstrate that the accumulation of ionic indium, but not ionic tin, from ITO NPs in the intracellular matrix has extensive cellular effects. PMID:26378248

  6. Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans

    PubMed Central

    Abegglen, Lisa M.; Caulin, Aleah F.; Chan, Ashley; Lee, Kristy; Robinson, Rosann; Campbell, Michael S.; Kiso, Wendy K.; Schmitt, Dennis L.; Waddell, Peter J; Bhaskara, Srividya; Jensen, Shane T.; Maley, Carlo C.; Schiffman, Joshua D.

    2016-01-01

    IMPORTANCE Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. OBJECTIVE To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). DESIGN, SETTING, AND PARTICIPANTS A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. EXPOSURES Ionizing radiation and doxorubicin. MAIN OUTCOMES AND MEASURES Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. RESULTS Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95%CI, 0%–5%]; African wild dog, 8%[95%CI, 0%–16%]; lion, 2%[95%CI, 0% –7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95%CI, 3.14%–6.49%), compared with humans, who have 11% to 25%cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain

  7. Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans

    DOE PAGES

    Abegglen, Lisa M.; Caulin, Aleah F.; Chan, Ashley; ...

    2015-10-08

    Here, evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). Design, Setting, and Participants A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n=644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro inmore » the laboratory for DNA damage response. The study included African and Asian elephants (n=8), patients with LFS (n=10), and age-matched human controls (n=11). Human samples were collected at the University of Utah between June 2014 and July 2015. Exposures Ionizing radiation and doxorubicin. Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes

  8. Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans

    SciTech Connect

    Abegglen, Lisa M.; Caulin, Aleah F.; Chan, Ashley; Lee, Kristy; Robinson, Rosann; Campbell, Michael S.; Kiso, Wendy K.; Schmitt, Dennis L.; Waddell, Peter J.; Bhaskara, Srividya; Jensen, Shane T.; Maley, Carlo C.; Schiffman, Joshua D.

    2015-10-08

    Here, evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). Design, Setting, and Participants A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n=644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n=8), patients with LFS (n=10), and age-matched human controls (n=11). Human samples were collected at the University of Utah between June 2014 and July 2015. Exposures Ionizing radiation and doxorubicin. Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes

  9. Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells

    PubMed Central

    Palma-Vera, Sergio E.; Schoen, Jennifer; Chen, Shuai

    2017-01-01

    Objective Ovarian steroid hormones (mainly E2 and P4) regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models. Methods A cell line derived from the porcine oviductal epithelium (CCLV-RIE270) was characterized (lineage markers, proliferation characteristics and transformation status). Primary porcine oviduct epithelial cells (POEC) were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml). Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR) and inflammation was quantified. Results CCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml) upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8). Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2. Conclusions CCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells. PMID:28231273

  10. Aberrant GLI1 Activation in DNA Damage Response, Carcinogenesis and Chemoresistance

    PubMed Central

    Palle, Komaraiah; Mani, Chinnadurai; Tripathi, Kaushlendra; Athar, Mohammad

    2015-01-01

    The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor β (TGFβ), wingless-type MMTV integration site family (WNT) and β-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for

  11. Hydrogen Bonding Motifs in MutSaphla and their response to binding damaged DNA

    NASA Astrophysics Data System (ADS)

    Negureanu, Lacra; Salsbury, Freddie

    2013-03-01

    Over the past decade, there has been a growing interest in studying the binding of damaged DNA to the MutSalpha protein complex. This protein complex, the Msh2/Msh6 complex in humans, is the initial complex that binds mismatched DNA and other DNA defects that occur during replication. This complex has also been shown to bind at least some types of damaged DNA. As a result of this interest, multiple studies have contrasted the interactions of MutSalpha with its normal mismatched substrate and with the interactions of MutsSalpha to DNA damaged by the chemotherapeutic cisplatin. To complement these studies, we examined the interaction between MutSalpha and DNA damaged by carboplatin via all-atom molecular dynamics simulations. These simulations provide evidence for different hydrogen bonding interactions at the protein/DNA and protein/protein interface. The hydrogen bonding motifs found are broadly similar to those found in binding to the adduct from cis-platin, but have distinct differences. These subtle differences may play a role in the way the different damages are signaled by MutS.

  12. Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication

    PubMed Central

    Zhao, Hong; Traganos, Frank; Darzynkiewicz, Zbigniew

    2010-01-01

    It has been reported that exposure to UV light triggers DNA damage response (DDR) seen as induction of γH2AX not only in S- but also in G1- phase cells. In the present study, in addition to γH2AX, we assessed other markers of DDR, namely phosphorylation of ATM on Ser1981, of ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains and of the tumor suppressor p53 on Ser15, in human pulmonary carcinoma A549 cells irradiated with 50 J/m2 of UV-B. Phosphorylation of these proteins detected with phospho-specific Abs and measured by laser scanning cytometry in relation the cell cycle phase was found to be selective to S-phase cells. The kinetics of phosphorylation of ATM was strikingly similar to that of ATM/ATR substrate, peaking at 30 min after UV irradiation and followed by rapid dephosphorylation. The peak of H2AX phosphorylation was seen at 2 h and the peak of p53 phosphorylation at 4 h after exposure to UV light. Local high spatial density of these phospho-proteins reported by intensity of maximal pixel of immunofluorescence in the DDR nuclear foci was distinctly more pronounced in the early compared to late portion of S-phase. Exposure of cells to UV following 1 h pulse-labeling of their DNA with 5-ethynyl-2′deoxyuridine (EdU) made it possible to correlate the extent of DNA replication during the pulse with the extent of the UV-induced H2AX phosphorylation within the same cells. This correlation was very strong (R2= 0.98) and the cells that did not incorporate EdU showed no evidence of H2AX phosphorylation. The data are consistent with the mechanism in which stalling of DNA replication forks upon collision with the primary UV-induced DNA lesions and likely formation of double-strand DNA breaks triggers DDR. The prior reports (including our own) on induction of γH2AX in G1 cells by UV may have erroneously identified cells initiating DNA replication following UV exposure as G1 cells due to the fact that their DNA content did not significantly differ from that of G

  13. TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling.

    PubMed

    Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji; An, You Sun; Yi, Jae Youn

    2016-08-05

    The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression.

  14. IFI16, an amplifier of DNA-damage response: Role in cellular senescence and aging-associated inflammatory diseases.

    PubMed

    Choubey, Divaker; Panchanathan, Ravichandran

    2016-07-01

    DNA-damage induces a DNA-damage response (DDR) in mammalian cells. The response, depending upon the cell-type and the extent of DNA-damage, ultimately results in cell death or cellular senescence. DDR-induced signaling in cells activates the ATM-p53 and ATM-IKKα/β-interferon (IFN)-β signaling pathways, thus leading to an induction of the p53 and IFN-inducible IFI16 gene. Further, upon DNA-damage, DNA accumulates in the cytoplasm, thereby inducing the IFI16 protein and STING-dependent IFN-β production and activation of the IFI16 inflammasome, resulting in the production of proinflammatory cytokines (e.g., IL-1β and IL-18). Increased expression of IFI16 protein in a variety of cell-types promotes cellular senescence. However, reduced expression of IFI16 in cells promotes cell proliferation. Because expression of the IFI16 gene is induced by activation of DNA-damage response in cells and increased levels of IFI16 protein in cells potentiate the p53-mediated transcriptional activation of genes and p53 and pRb-mediated cell cycle arrest, we discuss how an improved understanding of the role of IFI16 protein in cellular senescence and associated inflammatory secretory phenotype is likely to identify the molecular mechanisms that contribute to the development of aging-associated human inflammatory diseases and a failure to cancer therapy.

  15. DNA-damage response associated with occupational exposure, age and chronic inflammation in workers in the automotive industry.

    PubMed

    Savina, Natalya V; Smal, Marharyta P; Kuzhir, Tatyana D; Ershova-Pavlova, Alla A; Goncharova, Roza I

    2012-10-09

    The evaluation of genome integrity in populations occupationally exposed to combine industrial factors is of medical importance. In the present study, the DNA-damage response was estimated by means of the alkaline comet assay in a sizeable cohort of volunteers recruited among workers in the automotive industry. For this purpose, freshly collected lymphocytes were treated with hydrogen peroxide (100μM, 1min, 4°C) in vitro, and the levels of basal and H(2)O(2)-induced DNA damage, and the kinetics and efficiency of DNA repair were measured during a 180-min interval after exposure. The parameters studied in the total cohort of workers were in a range of values prescribed for healthy adult residents of Belarus. Based on the 95th percentiles, individuals possessing enhanced cellular sensitivity to DNA damage were present in different groups, but the frequency was significantly higher among elderly persons and among individuals with chronic inflammatory diseases. The results indicate that the inter-individual variations in DNA-damage response should be taken into account to estimate adequately the environmental genotoxic effects and to identify individuals with an enhanced DNA-damage response due to the influence of some external factors or intrinsic properties of the organism. Underling mechanisms need to be further explored.

  16. EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele

    PubMed Central

    Rein, Katrin; Yanez, Diana A.; Terré, Berta; Palenzuela, Lluís; Aivio, Suvi; Wei, Kaichun; Edelmann, Winfried; Stark, Jeremy M.; Stracker, Travis H.

    2015-01-01

    The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5′-3′ exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations. PMID:26160886

  17. Down-regulation of histone H2B by DNA-dependent protein kinase in response to DNA damage through modulation of octamer transcription factor 1.

    PubMed

    Schild-Poulter, Caroline; Shih, Amy; Yarymowich, Nicholas C; Haché, Robert J G

    2003-11-01

    Cells respond to double-stranded DNA breaks (DSBs) by pausing cell cycle progression to allow the repair machinery to restore genomic integrity. DNA-dependent protein kinase (DNA-PK), comprising a large catalytic subunit (DNA-PK(cs)) and the Ku antigen regulatory subunit (Ku70/Ku80), is activated in response to DSBs and is required for DNA repair through the nonhomologous end-joining pathway. Here we provide evidence that DNA-PK participates in altering specific gene expression in response to DNA damage by modulating the stability and transcriptional regulatory potential of the essential transcription factor octamer transcription factor 1 (Oct-1). Histone H2B and U2 RNA, whose expression are highly dependent on Oct-1, were strongly decreased in response to ionizing radiation in a DNA-PK-dependent manner, and Oct-1-dependent reporter gene transcription was repressed. Furthermore, Oct-1 phosphorylation in response to ionizing radiation increased in a DNA-PK-dependent manner. Paradoxically, down-regulation of transactivation correlated with the rapid DNA-PK-dependent stabilization of Oct-1. Stabilization of Oct-1 was dependent on the NH(2)-terminal region of Oct-1, which contains a transcriptional activation domain and which was phosphorylated by DNA-PK in vitro. These results suggest a mechanism for the regulation of Oct-1 in response to DNA damage through specific phosphorylation within the NH(2)-terminal transcriptional regulatory domain.

  18. Population variability in biological adaptive responses to DNA damage and the shapes of carcinogen dose-response curves

    SciTech Connect

    Conolly, Rory B. . E-mail: Conolly.Rory@epa.gov; Gaylor, David W.; Lutz, Werner K.

    2005-09-01

    Carcinogen dose-response curves for both ionizing radiation and chemicals are typically assumed to be linear at environmentally relevant doses. This assumption is used to ensure protection of the public health in the absence of relevant dose-response data. A theoretical justification for the assumption has been provided by the argument that low dose linearity is expected when an exogenous agent adds to an ongoing endogenous process. Here, we use computational modeling to evaluate (1) how two biological adaptive processes, induction of DNA repair and cell cycle checkpoint control, may affect the shapes of dose-response curves for DNA-damaging carcinogens and (2) how the resulting dose-response behaviors may vary within a population. Each model incorporating an adaptive process was capable of generating not only monotonic dose-responses but also nonmonotonic (J-shaped) and threshold responses. Monte Carlo analysis suggested that all these dose-response behaviors could coexist within a population, as the spectrum of qualitative differences arose from quantitative changes in parameter values. While this analysis is largely theoretical, it suggests that (a) accurate prediction of the qualitative form of the dose-response requires a quantitative understanding of the mechanism (b) significant uncertainty is associated with human health risk prediction in the absence of such quantitative understanding and (c) a stronger experimental and regulatory focus on biological mechanisms and interindividual variability would allow flexibility in regulatory treatment of environmental carcinogens without compromising human health.

  19. Breast Cancer–Associated Abraxas Mutation Disrupts Nuclear Localization and DNA Damage Response Functions

    PubMed Central

    Solyom, Szilvia; Aressy, Bernadette; Pylkäs, Katri; Patterson-Fortin, Jeffrey; Hartikainen, Jaana M.; Kallioniemi, Anne; Kauppila, Saila; Nikkilä, Jenni; Kosma, Veli-Matti; Mannermaa, Arto; Greenberg, Roger A.; Winqvist, Robert

    2013-01-01

    Breast cancer is the most common cancer in women in developed countries and has a well-established genetic component. Germline mutations in a network of genes encoding BRCA1, BRCA2, and their interacting partners confer hereditary susceptibility to breast cancer. Abraxas directly interacts with the BRCA1 BRCT (BRCA1 carboxyl-terminal) repeats and contributes to BRCA1-dependent DNA damage responses, making Abraxas a candidate for yet unexplained disease susceptibility. Here, we have screened 125 Northern Finnish breast cancer families for coding region and splice-site Abraxas mutations and genotyped three tagging single-nucleotide polymorphisms within the gene from 991 unselected breast cancer cases and 868 female controls for common cancer-associated variants. A novel heterozygous alteration, c.1082G>A (Arg361Gln), that results in abrogated nuclear localization and DNA response activities was identified in three breast cancer families and in one additional familial case from an unselected breast cancer cohort, but not in healthy controls (P = 0.002). On the basis of its exclusive occurrence in familial cancers, disease cosegregation, evolutionary conservation, and disruption of critical BRCA1 functions, the recurrent Abraxas c.1082G>A mutation connects to cancer predisposition. These findings contribute to the concept of a BRCA-centered tumor suppressor network and provide the identity of Abraxas as a new breast cancer susceptibility gene. PMID:22357538

  20. USP45 deubiquitylase controls ERCC1–XPF endonuclease-mediated DNA damage responses

    PubMed Central

    Perez-Oliva, Ana B; Lachaud, Christophe; Szyniarowski, Piotr; Muñoz, Ivan; Macartney, Thomas; Hickson, Ian; Rouse, John; Alessi, Dario R

    2015-01-01

    Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF–ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1–XPF. Together, these results establish USP45 as a new regulator of XPF–ERCC1 crucial for efficient DNA repair. PMID:25538220

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

    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 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus 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 profiles 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 γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not

  2. DNA Damage and Its Cellular Response in Mother and Fetus Exposed to Hyperglycemic Environment

    PubMed Central

    Moreli, Jusciele Brogin; Santos, Janine Hertzog; Rocha, Clarissa Ribeiro; Rudge, Marilza Vieira; Bevilacqua, Estela; Calderon, Iracema Mattos Paranhos

    2014-01-01

    The increased production of reactive oxygen species (ROS) plays a key role in pathogenesis of diabetic complications. ROS are generated by exogenous and endogenous factors such as during hyperglycemia. When ROS production exceeds the detoxification and scavenging capacity of the cell, oxidative stress ensues. Oxidative stress induces DNA damage and when DNA damage exceeds the cellular capacity to repair it, the accumulation of errors can overwhelm the cell resulting in cell death or fixation of genome mutations that can be transmitted to future cell generations. These mutations can lead to and/or play a role in cancer development. This review aims at (i) understanding the types and consequences of DNA damage during hyperglycemic pregnancy; (ii) identifying the biological role of DNA repair during pregnancy, and (iii) proposing clinical interventions to maintain genome integrity. While hyperglycemia can damage the maternal genetic material, the impact of hyperglycemia on fetal cells is still unclear. DNA repair mechanisms may be important to prevent the deleterious effects of hyperglycemia both in mother and in fetus DNA and, as such, prevent the development of diseases in adulthood. Hence, in clinical practice, maternal glycemic control may represent an important point of intervention to prevent the deleterious effects of maternal hyperglycemia to DNA. PMID:25197655

  3. GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

    2015-01-01

    Background. Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis. PMID:26273425

  4. NF-κB-HOTAIR axis links DNA damage response, chemoresistance and cellular senescence in ovarian cancer

    PubMed Central

    Özeş, Ali R.; Miller, David F.; Özeş, Osman N.; Fang, Fang; Liu, Yunlong; Matei, Daniela; Huang, Tim; Nephew, Kenneth P.

    2016-01-01

    The transcription factor nuclear factor kappa B (NF-κB) and the long non-coding RNA (lncRNA) HOTAIR (HOX transcript antisense RNA) play diverse functional roles in cancer. In this study, we show that upregulation of HOTAIR induced platinum resistance in ovarian cancer, and increased HOTAIR levels were observed in recurrent platinum-resistant ovarian tumors vs. primary ovarian tumors. To investigate the role of HOTAIR during DNA damage induced by platinum, we monitored double-strand breaks and show that HOTAIR expression results in sustained activation of DNA damage response after platinum treatment. We demonstrate that ectopic expression of HOTAIR induces NF-κB activation during DNA damage response and MMP-9 and IL-6 expression, both key NF-κB target genes. We show that HOTAIR regulates activation of NF-κB by decreasing Iκ-Bα (NF-κB inhibitor) and establish that by inducing prolonged NF-κB activation and expression of NF-κB target genes during DNA damage, HOTAIR plays a critical role in cellular senescence and platinum sensitivity. Our findings suggest that a NF-κB-HOTAIR axis drives a positive-feedback loop cascade during DNA damage response and contributes to cellular senescence and chemotherapy resistance in ovarian and other cancers. PMID:27041570

  5. Effects of antidepressants on DSP4/CPT-induced DNA damage response in neuroblastoma SH-SY5Y cells

    PubMed Central

    Wang, Yan; Hilton, Benjamin A.; Cui, Kui; Zhu, Meng-Yang

    2015-01-01

    DNA damage is a form of cell stress and injury. Increased systemic DNA damage is related to the pathogenic development of neurodegenerative diseases. Depression occurs in a relatively high percentage of patients suffering from degenerative diseases, for whom antidepressants are often used to relieve depressive symptoms. However, few studies have attempted to elucidate why different groups of antidepressants have similar effects on relieving symptoms of depression. Previously, we demonstrated that neurotoxins N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4)- and camptothecin (CPT)-induced the DNA damage response in SH-SY5Y cells, and DSP4 caused cell cycle arrest which was predominately in the S-phase. The present study shows that CPT treatment also resulted in similar cell cycle arrest. Some classic antidepressants could reduce the DNA damage response induced by DSP4 or CPT in SH-SY5Y cells. Cell viability examination demonstrated that both DSP4 and CPT caused cell death, which was prevented by spontaneous administration of some tested antidepressants. Flow cytometric analysis demonstrated that a majority of the tested antidepressants protect cells from being arrested in S-phase. These results suggest that blocking the DNA damage response may be an important pharmacologic characteristic of antidepressants. Exploring the underlying mechanisms may allow for advances in the effort to improve therapeutic strategies for depression appearing in degenerative and psychiatric diseases. PMID:26038195

  6. Conversion of DNA damage into chromosome damage in response to cell cycle regulation of chromatin condensation after irradiation.

    PubMed

    Terzoudi, G I; Pantelias, G E

    1997-07-01

    Cell fusion, premature chromosome condensation (PCC) and conventional cytogenetics were used to test whether the biochemical process of chromatin condensation-decondensation throughout the cell cycle, which depends on cyclin-regulated histone H1 kinase activity, affects the conversion of DNA damage into chromosome damage and determines intrinsic cell cycle-stage radiosensitivity. Results from three sets of experiments are presented. Irradiated G0 human lymphocytes were fused to exponentially growing hamster cells and time allowed for repair, while following the hamster cells in their progress towards mitosis. Severe fragmentation was observed in the induced lymphocyte PCCs when hamster cells entered mitosis 13 h after irradiation, suggesting conversion of DNA damage into non-repairable chromosome damage during G1/S transition. When PCC was used to analyse chromosome damage directly in G0 and G2 phase lymphocytes, the induction of breaks per cell per chromatid per Gy was found to be similar, suggesting that G2 increased radiosensitivity is related to chromatin condensation occurring during G2/M transition and not to an inherent chromatin structure at this phase. When chromatin condensation-decondensation at the G1/S and G2/M transitions was modified after irradiation by using conditioned media or elevated temperature (40 degrees C), a dramatic change in the yield and the type of chromosomal aberrations was observed. All results obtained were consistent with the proposed hypothesis. They may be also helpful in the characterization of a DNA-chromosome damage conversion process which could give a biochemical explanation of the variability in radiosensitivity observed at the various stages of the cell cycle as well as among mutant cells and cells of different origin. The proposed conversion process is cell cycle-regulated and, therefore, subject to up-regulation or down-regulation following mutagen exposure and genetic alterations.

  7. p27kip1 stabilization is essential for the maintenance of cell cycle arrest in response to DNA damage

    PubMed Central

    Cuadrado, Myriam; Gutierrez-Martinez, Paula; Swat, Aneta; Nebreda, Angel R.; Fernandez-Capetillo, Oscar

    2013-01-01

    One of the current models of cancer proposes that oncogenes activate a DNA damage response (DDR), which would limit the growth of the tumor in its earliest stages. In this context, and in contrast to studies focused on the acute responses to a one-time genotoxic insult, understanding how cells respond to a persistent source of DNA damage might become critical for future studies in the field. We here report the discovery of a novel damage-responsive pathway, which involves p27kip1 and retinoblastoma tumour suppressors (TS), and which is only implemented after a persistent exposure to clastogens. In agreement with its late activation, we show that this pathway is critical for the maintenance –but not the initiation- of the cell cycle arrest triggered by DNA damage. Interestingly, this late response is independent of the canonical ATM- and ATR-dependent DDR, but downstream of p38 MAPK. Our results might help to reconcile the oncogene-induced DNA damage model with the clinical evidence that points to non-DDR members as the most important TSs in human cancer. PMID:19843869

  8. Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

    SciTech Connect

    Kiran, Shashi; Oddi, Vineesha; Ramakrishna, Gayatri

    2015-02-01

    Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

  9. DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer.

    PubMed

    Sears, Catherine R; Cooney, Sean A; Chin-Sinex, Helen; Mendonca, Marc S; Turchi, John J

    2016-04-01

    Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24h after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism.

  10. DNA damage response is hijacked by human papillomaviruses to complete their life cycle.

    PubMed

    Hong, Shi-Yuan

    The DNA damage response (DDR) is activated when DNA is altered by intrinsic or extrinsic agents. This pathway is a complex signaling network and plays important roles in genome stability, tumor transformation, and cell cycle regulation. Human papillomaviruses (HPVs) are the main etiological agents of cervical cancer. Cervical cancer ranks as the fourth most common cancer among women and the second most frequent cause of cancer-related death worldwide. Over 200 types of HPVs have been identified and about one third of these infect the genital tract. The HPV life cycle is associated with epithelial differentiation. Recent studies have shown that HPVs deregulate the DDR to achieve a productive life cycle. In this review, I summarize current findings about how HPVs mediate the ataxia-telangiectasia mutated kinase (ATM) and the ATM-and RAD3-related kinase (ATR) DDRs, and focus on the roles that ATM and ATR signalings play in HPV viral replication. In addition, I demonstrate that the signal transducer and activator of transcription-5 (STAT)-5, an important immune regulator, can promote ATM and ATR activations through different mechanisms. These findings may provide novel opportunities for development of new therapeutic targets for HPV-related cancers.

  11. A Novel Aspect of Tumorigenesis-BMI1 Functions in Regulating DNA Damage Response.

    PubMed

    Lin, Xiaozeng; Ojo, Diane; Wei, Fengxiang; Wong, Nicholas; Gu, Yan; Tang, Damu

    2015-12-01

    BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs) for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT). Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1), and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub), BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR). In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs) through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis.

  12. DNA damage response is hijacked by human papillomaviruses to complete their life cycle

    PubMed Central

    Hong, Shi-yuan

    2017-01-01

    The DNA damage response (DDR) is activated when DNA is altered by intrinsic or extrinsic agents. This pathway is a complex signaling network and plays important roles in genome stability, tumor transformation, and cell cycle regulation. Human papillomaviruses (HPVs) are the main etiological agents of cervical cancer. Cervical cancer ranks as the fourth most common cancer among women and the second most frequent cause of cancer-related death worldwide. Over 200 types of HPVs have been identified and about one third of these infect the genital tract. The HPV life cycle is associated with epithelial differentiation. Recent studies have shown that HPVs deregulate the DDR to achieve a productive life cycle. In this review, I summarize current findings about how HPVs mediate the ataxia-telangiectasia mutated kinase (ATM) and the ATM-and RAD3-related kinase (ATR) DDRs, and focus on the roles that ATM and ATR signalings play in HPV viral replication. In addition, I demonstrate that the signal transducer and activator of transcription-5 (STAT)-5, an important immune regulator, can promote ATM and ATR activations through different mechanisms. These findings may provide novel opportunities for development of new therapeutic targets for HPV-related cancers. PMID:28271657

  13. An alternative telomerase RNA in Arabidopsis modulates enzyme activity in response to DNA damage

    PubMed Central

    Cifuentes-Rojas, Catherine; Nelson, Andrew D.L.; Boltz, Kara A.; Kannan, Kalpana; She, Xintao; Shippen, Dorothy E.

    2012-01-01

    Telomerase replenishes telomere tracts by reiteratively copying its RNA template, TER. Unlike other model organisms, Arabidopsis thaliana harbors two divergent TER genes. However, only TER1 is required for telomere maintenance. Here we examine the function of TER2. We show that TER2 is spliced and its 3′ end is truncated in vivo to generate a third TER isoform, TER2S. TERT preferentially associates with TER2 > TER1 > TER2S. Moreover, TER2 and TER2S assemble with Ku and POT1b (protection of telomeres), forming RNP (ribonucleoprotein) complexes distinct from TER1 RNP. Plants null for TER2 display increased telomerase enzyme activity, while TER2 overexpression inhibits telomere synthesis from TER1 and leads to telomere shortening. These findings argue that TER2 negatively regulates telomerase by sequestering TERT in a nonproductive RNP complex. Introduction of DNA double-strand breaks by zeocin leads to an immediate and specific spike in TER2 and a concomitant decrease in telomerase enzyme activity. This response is not triggered by replication stress or telomere dysfunction and is abrogated in ter2 mutants. We conclude that Arabidopsis telomerase is modulated by TER2, a novel DNA damage-induced noncoding RNA that works in concert with the canonical TER to promote genome integrity. PMID:23109676

  14. MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

    SciTech Connect

    Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala; Glas, Rickard

    2010-08-27

    Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

  15. MASTL(Greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation

    PubMed Central

    Wong, Po Yee; Ma, Hoi Tang; Lee, Hyun-jung; Poon, Randy Y. C.

    2016-01-01

    The G2 DNA damage checkpoint is one of the most important mechanisms controlling G2–mitosis transition. The kinase Greatwall (MASTL in human) promotes normal G2–mitosis transition by inhibiting PP2A via ARPP19 and ENSA. In this study, we demonstrate that MASTL is critical for maintaining genome integrity after DNA damage. Although MASTL did not affect the activation of DNA damage responses and subsequent repair, it determined the timing of entry into mitosis and the subsequent fate of the recovering cells. Constitutively active MASTL promoted dephosphorylation of CDK1Tyr15 and accelerated mitotic entry after DNA damage. Conversely, downregulation of MASTL or ARPP19/ENSA delayed mitotic entry. Remarkably, APC/C was activated precociously, resulting in the damaged cells progressing from G2 directly to G1 and skipping mitosis all together. Collectively, these results established that precise control of MASTL is essential to couple DNA damage to mitosis through the rate of mitotic entry and APC/C activation. PMID:26923777

  16. MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response.

    PubMed

    Staples, Christopher J; Barone, Giancarlo; Myers, Katie N; Ganesh, Anil; Gibbs-Seymour, Ian; Patil, Abhijit A; Beveridge, Ryan D; Daye, Caroline; Beniston, Richard; Maslen, Sarah; Ahel, Ivan; Skehel, J Mark; Collis, Spencer J

    2016-09-06

    Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN) damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP). We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP's role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response.

  17. Induction of DNA Damage Response by the Supravital Probes of Nucleic Acids

    PubMed Central

    Zhao, Hong; Traganos, Frank; Dobrucki, Jurek; Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2009-01-01

    The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV) and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981) and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2 or p53 phosphorylation. However, pronounced ATM, Chk2 and p53 phosphorylation and perturbed G2 progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2 and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2 and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G2M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5 and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis. PMID:19373929

  18. Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

    PubMed

    Phesse, T J; Myant, K B; Cole, A M; Ridgway, R A; Pearson, H; Muncan, V; van den Brink, G R; Vousden, K H; Sears, R; Vassilev, L T; Clarke, A R; Sansom, O J

    2014-06-01

    Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

  19. A Fhit-ing role in the DNA damage checkpoint response.

    PubMed

    Ishii, Hideshi; Wang, Ya; Huebner, Kay

    2007-05-02

    The FHIT gene encompasses the most active common fragile site of the human genome and is thus exquisitely sensitive to intragenic alterations by DNA damaging agents, alterations that can lead to FHIT allele loss very early in the preneoplastic phase of cancer development, before or coincident with activation of the DNA damage checkpoint. Fhit protein expression is lost or reduced in many preneoplastic lesions and in >50% of cancers, Fhit knockout mice are highly susceptible to carcinogen induction of tumors and Fhit replacement in these mice by gene therapy induces apoptosis and significantly reduces tumor burden. But learning how Fhit induces apoptosis and suppresses tumors has been a challenge because interacting proteins, effectors of Fhit signals, have not been discovered. Nevertheless, the study of Fhit deficient mouse and human tissue-derived and cancer-derived cells in vitro has led to several important conclusions: repair protein-deficient cancers are more likely to be Fhit-deficient; Fhit-deficient cells show enhanced resistance to UVC, mitomycin C, camptothecin and ionizing radiation-induced cell killing, possibly due to strong activation of the ATR pathway following DNA damage; Fhit-deficient cells show higher efficiency of homologous recombination repair, a double-strand break repair pathway in mammalian cells; Fhit protein indirectly affects S-phase checkpoint and DNA repair. Finally, results of a recent study have suggested that the DNA damage-susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein, Fhit, that is necessary for protecting cells from accumulation of DNA damage, through modulation of checkpoint proteins Hus1 and phosphoChk1. Thus, inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. It will be very important to determine mechanisms employed by Fhit in modulating checkpoint pathways, and to define consequences of Fhit loss in specific preneoplastic and

  20. Concepts of Chronic Lymphocytic Leukemia Pathogenesis: DNA Damage Response and Tumor Microenvironment.

    PubMed

    Frenzel, Lukas P; Reinhardt, H Christian; Pallasch, Christian P

    2016-01-01

    Pathogenesis of chronic lymphocytic leukemia (CLL) is characterized by specific genetic aberrations and alterations of cellular signaling pathways. In particular, a disturbed DNA damage response (DDR) and an activated B-cell receptor signaling pathway play a major role in promoting CLL cell survival. External stimuli are similarly essential for CLL cell survival and lead to activation of the PI3K/AKT and MAPK pathways. Activation of nuclear factor-kappa B (NFkB) influences the disturbed anti-apoptotic balance of CLL cells. Losses or disabling mutations in TP53 and ATM are frequent events in chemotherapy-naïve patients and are further enriched in chemotherapy-resistant patients. As these lesions define key regulatory elements of the DDR pathway, they also determine treatment response to genotoxic therapy. Novel therapeutic strategies therefore try to circumvent defective DDR signaling and to suppress the pro-survival stimuli received from the tumor microenvironment. With increasing knowledge on specific genetic alterations of CLL, we may be able to target CLL cells more efficiently even in the situation of mutated DDR pathways or protection by microenvironmental stimuli.

  1. Oxidative stress and DNA damage responses to phenanthrene exposure in the estuarine guppy Poecilia vivipara.

    PubMed

    Machado, Anderson Abel de Souza; Hoff, Mariana Leivas Müller; Klein, Roberta Daniele; Cordeiro, Gilson Junior; Lencina Avila, Jannine Marquez; Costa, Patrícia Gomes; Bianchini, Adalto

    2014-07-01

    Despite ubiquitous phenanthrene contamination in aquatic coastal areas, little is known regarding its potential effects on estuarine fishes. The present work evaluated the response of a large suite of oxidative stress- and DNA damage-related biomarkers to phenanthrene exposure (10, 20 and 200 μg L(-1), 96 h) using DMSO as the solvent in estuarine guppy Poecilia vivipara (salinity 24 psu). Phenanthrene affected oxidative stress-related parameters, and decreased antioxidant defenses and reactive oxygen species in the gills and muscle overall. Lipid peroxidation occurred in muscle at 200 μg L(-1) phenanthrene. Genotoxicity was increased at 20 μg L(-1), while 200 μg L(-1) caused a relative decrease in erythrocyte release into the bloodstream. These findings indicated that phenanthrene is genotoxic and can induce oxidative stress, depending on tissue and phenanthrene concentration analyzed. Thus, some of the biomarkers analyzed in the present study are sufficiently sensitive to monitor the exposure of the guppy P. vivipara to phenanthrene in salt water. However, further studies are required for a better interpretation of the dose-response patterns observed.

  2. Activation of DNA Damage Response Induced by the Kaposi's Sarcoma-Associated Herpes Virus.

    PubMed

    Di Domenico, Enea Gino; Toma, Luigi; Bordignon, Valentina; Trento, Elisabetta; D'Agosto, Giovanna; Cordiali-Fei, Paola; Ensoli, Fabrizio

    2016-06-01

    The human herpes virus 8 (HHV-8), also known as Kaposi sarcoma-associated herpes virus (KSHV), can infect endothelial cells often leading to cell transformation and to the development of tumors, namely Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and the plasmablastic variant of multicentric Castleman's disease. KSHV is prevalent in areas such as sub-Saharan Africa and the Mediterranean region presenting distinct genotypes, which appear to be associated with differences in disease manifestation, according to geographical areas. In infected cells, KSHV persists in a latent episomal form. However, in a limited number of cells, it undergoes spontaneous lytic reactivation to ensure the production of new virions. During both the latent and the lytic cycle, KSHV is programmed to express genes which selectively modulate the DNA damage response (DDR) through the activation of the ataxia telangiectasia mutated (ATM) pathway and by phosphorylating factors associated with the DDR, including the major tumor suppressor protein p53 tumor suppressor p53. This review will focus on the interplay between the KSHV and the DDR response pathway throughout the viral lifecycle, exploring the putative molecular mechanism/s that may contribute to malignant transformation of host cells.

  3. DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

    PubMed Central

    Mimmler, Maximilian; Peter, Simon; Kraus, Alexander; Stroh, Svenja; Nikolova, Teodora; Seiwert, Nina; Hasselwander, Solveig; Neitzel, Carina; Haub, Jessica; Monien, Bernhard H.; Nicken, Petra; Steinberg, Pablo; Shay, Jerry W.; Kaina, Bernd; Fahrer, Jörg

    2016-01-01

    PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs. PMID:27599846

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

    EPA Science Inventory

    Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

  5. Common cancer-associated imbalances in the DNA damage response confer sensitivity to single agent ATR inhibition

    PubMed Central

    Middleton, Fiona K.; Patterson, Miranda J.; Elstob, Claire J.; Fordham, Sarah; Herriott, Ashleigh; Wade, Mark A.; McCormick, Aiste; Edmondson, Richard; May, Felicity E.B.; Allan, James M.; Pollard, John R.; Curtin, Nicola J.

    2015-01-01

    ATRis an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest, DNA damage accumulation and repair were determined following VE-821 exposure. Defectsin homologous recombination repair (HRR: ATM, BRCA2 and XRCC3) and baseexcision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly, the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component, Ku80, caused hypersensitivity to VE-821, but loss of its partner catalytic subunit, DNA-PKcs, did not. Unexpectedly, VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR, determined by RAD51 focus formation, to a greater extent in cells with high DNA-PKcs. Defects in HRR and BER and high DNA-PKcs expression, that are common in cancer, confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine. PMID:26486089

  6. Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.

    PubMed

    Escarceller, M; Hicks, J; Gudmundsson, G; Trump, G; Touati, D; Lovett, S; Foster, P L; McEntee, K; Goodman, M F

    1994-10-01

    DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.

  7. The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response.

    PubMed

    Brestovitsky, Anna; Nebenzahl-Sharon, Keren; Kechker, Peter; Sharf, Rakefet; Kleinberger, Tamar

    2016-02-01

    The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition

  8. The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

    PubMed Central

    Kechker, Peter; Sharf, Rakefet; Kleinberger, Tamar

    2016-01-01

    The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition

  9. Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM.

    PubMed

    McCabe, Nuala; Hanna, Conor; Walker, Steven M; Gonda, David; Li, Jie; Wikstrom, Katarina; Savage, Kienan I; Butterworth, Karl T; Chen, Clark; Harkin, D Paul; Prise, Kevin M; Kennedy, Richard D

    2015-06-01

    Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors.

  10. CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

    PubMed Central

    Becherel, Olivier J.; Jakob, Burkhard; Cherry, Amy L.; Gueven, Nuri; Fusser, Markus; Kijas, Amanda W.; Peng, Cheng; Katyal, Sachin; McKinnon, Peter J.; Chen, Junjie; Epe, Bernd; Smerdon, Stephen J.; Taucher-Scholz, Gisela; Lavin, Martin F.

    2010-01-01

    Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1. PMID:20008512

  11. Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells.

    PubMed

    Tan, Kang Wei; Pham, Tuan Minh; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

    2015-02-18

    The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30-50% in E. coli cells, leading to a 20-30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response.

  12. NFBD1/MDC1 stabilizes oncogenic MDM2 to contribute to cell fate determination in response to DNA damage

    SciTech Connect

    Inoue, Ken-ichi; Nakanjishi, Mitsuru; Kikuchi, Hironobu; Yamamoto, Hideki; Todo, Satoru; Nakagawara, Akira Ozaki, Toshinori

    2008-07-11

    In response to DNA damage, NFBD1/MDC1 induces the accumulation of DNA repair machinery such as MRN complex at the sites of damaged DNA to form nuclear foci. In this study, we found that NFBD1 directly interacts with MDM2 and increases its stability. During adriamycin (ADR)-mediated apoptosis, expression levels of NFBD1 reduced in association with the down-regulation of MDM2. Enforced expression of NFBD1 resulted in a significant stabilization of MDM2. Consistent with these observations, siRNA-mediated knockdown of the endogenous NFBD1 decreased the amounts of the endogenous MDM2. Immunoprecipitation and in vitro pull-down assays demonstrated that NFBD1 interacts with MDM2 through its COOH-terminal BRCT domains. In accordance with our recent results, enforced expression of NFBD1 rendered cells resistant to DNA damage. Similar results were also obtained in cells expressing exogenous MDM2. Taken together, our present findings suggest that NFBD1-mediated stabilization contributes to cell survival in response to DNA damage.

  13. p53 pathway determines the cellular response to alcohol-induced DNA damage in MCF-7 breast cancer cells

    PubMed Central

    Zhao, Ming; Howard, Erin W.; Guo, Zhiying; Parris, Amanda B.; Yang, Xiaohe

    2017-01-01

    Alcohol consumption is associated with increased breast cancer risk; however, the underlying mechanisms that contribute to mammary tumor initiation and progression are unclear. Alcohol is known to induce oxidative stress and DNA damage; likewise, p53 is a critical modulator of the DNA repair pathway and ensures genomic integrity. p53 mutations are frequently detected in breast and other tumors. The impact of alcohol on p53 is recognized, yet the role of p53 in alcohol-induced mammary carcinogenesis remains poorly defined. In our study, we measured alcohol-mediated oxidative DNA damage in MCF-7 cells using 8-OHdG and p-H2AX foci formation assays. p53 activity and target gene expression after alcohol exposure were determined using p53 luciferase reporter assay, qPCR, and Western blotting. A mechanistic study delineating the role of p53 in DNA damage response and cell cycle arrest was based on isogenic MCF-7 cells stably transfected with control (MCF-7/Con) or p53-targeting siRNA (MCF-7/sip53), and MCF-7 cells that were pretreated with Nutlin-3 (Mdm2 inhibitor) to stabilize p53. Alcohol treatment resulted in significant DNA damage in MCF-7 cells, as indicated by increased levels of 8-OHdG and p-H2AX foci number. A p53-dependent signaling cascade was stimulated by alcohol-induced DNA damage. Moderate to high concentrations of alcohol (0.1–0.8% v/v) induced p53 activation, as indicated by increased p53 phosphorylation, reporter gene activity, and p21/Bax gene expression, which led to G0/G1 cell cycle arrest. Importantly, compared to MCF-7/Con cells, alcohol-induced DNA damage was significantly enhanced, while alcohol-induced p21/Bax expression and cell cycle arrest were attenuated in MCF-7/sip53 cells. In contrast, inhibition of p53 degradation via Nutlin-3 reinforced G0/G1 cell cycle arrest in MCF-7 control cells. Our study suggests that functional p53 plays a critical role in cellular responses to alcohol-induced DNA damage, which protects the cells from DNA damage

  14. A Novel Aspect of Tumorigenesis—BMI1 Functions in Regulating DNA Damage Response

    PubMed Central

    Lin, Xiaozeng; Ojo, Diane; Wei, Fengxiang; Wong, Nicholas; Gu, Yan; Tang, Damu

    2015-01-01

    BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs) for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT). Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1), and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub), BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR). In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs) through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis. PMID:26633535

  15. WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response.

    PubMed

    Abu-Odeh, Mohammad; Salah, Zaidoun; Herbel, Christoph; Hofmann, Thomas G; Aqeilan, Rami I

    2014-11-04

    Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.

  16. WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response

    PubMed Central

    Abu-Odeh, Mohammad; Salah, Zaidoun; Herbel, Christoph; Hofmann, Thomas G.; Aqeilan, Rami I.

    2014-01-01

    Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells. PMID:25331887

  17. Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    The RNR2 gene encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of the deoxyribonucleotides needed for DNA synthesis. Transcription of this gene is induced approximately 20-fold in response to environmental stimuli that damage DNA or block DNA replication. Deletion and subcloning analysis identified two, and possibly three, upstream activating sequences (UAS) and one repressing (URS) element in the RNR2 regulatory region. A 42-base-pair (bp) fragment from this region was found to be necessary for proper regulation of RNR2 and to be capable of conferring DNA damage inducibility upon a heterologous promoter. This fragment contained both positively and negatively acting sequences. Four DNA-binding factors interacted with the RNR2 regulatory region. One factor was identified as the GRF1 protein, the product of the RAP1 gene. GRF1 bound to the UAS2 element of RNR2, which was found to be directly adjacent to the 42-bp fragment. UAS2 activity was repressed by the 42-bp fragment. Three other factors bound to the 42-bp fragment; one of these factors, RRF3, had a second binding site in the RNR2 promoter. These factors are likely to mediate the response of RNR2 to DNA damage. Images PMID:2685561

  18. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis.

    PubMed

    Schmitt, Estelle; Paquet, Claudie; Beauchemin, Myriam; Bertrand, Richard

    2007-06-01

    Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senescence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent. Extensive research on tumor suppressor genes, oncogenes, the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are tied to cell proliferation, cell-cycle arrest, cellular senescence and apoptosis. DNA-damage responses are complex, involving "sensor" proteins that sense the damage, and transmit signals to "transducer" proteins, which, in turn, convey the signals to numerous "effector" proteins implicated in specific cellular pathways, including DNA repair mechanisms, cell-cycle checkpoints, cellular senescence and apoptosis. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/recombination and cellular senescence, effects that are generally distinct from their function in apoptosis. In this review, we report progress in understanding the molecular networks that regulate cell-cycle checkpoints, cellular senescence and apoptosis after DNA damage, and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

  19. Tumor Suppressor Genes within Common Fragile Sites Are Active Players in the DNA Damage Response.

    PubMed

    Hazan, Idit; Hofmann, Thomas G; Aqeilan, Rami I

    2016-12-01

    The role of common fragile sites (CFSs) in cancer remains controversial. Two main views dominate the discussion: one suggests that CFS loci are hotspots of genomic instability leading to inactivation of genes encoded within them, while the other view proposes that CFSs are functional units and that loss of the encoded genes confers selective pressure, leading to cancer development. The latter view is supported by emerging evidence showing that expression of a given CFS is associated with genome integrity and that inactivation of CFS-resident tumor suppressor genes leads to dysregulation of the DNA damage response (DDR) and increased genomic instability. These two viewpoints of CFS function are not mutually exclusive but rather coexist; when breaks at CFSs are not repaired accurately, this can lead to deletions by which cells acquire growth advantage because of loss of tumor suppressor activities. Here, we review recent advances linking some CFS gene products with the DDR, genomic instability, and carcinogenesis and discuss how their inactivation might represent a selective advantage for cancer cells.

  20. Taming the tiger by the tail: modulation of DNA damage responses by telomeres.

    PubMed

    Lydall, David

    2009-08-05

    Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell-cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell-cycle division are discussed.

  1. Tumor Suppressor Genes within Common Fragile Sites Are Active Players in the DNA Damage Response

    PubMed Central

    Hazan, Idit; Hofmann, Thomas G.; Aqeilan, Rami I.

    2016-01-01

    The role of common fragile sites (CFSs) in cancer remains controversial. Two main views dominate the discussion: one suggests that CFS loci are hotspots of genomic instability leading to inactivation of genes encoded within them, while the other view proposes that CFSs are functional units and that loss of the encoded genes confers selective pressure, leading to cancer development. The latter view is supported by emerging evidence showing that expression of a given CFS is associated with genome integrity and that inactivation of CFS-resident tumor suppressor genes leads to dysregulation of the DNA damage response (DDR) and increased genomic instability. These two viewpoints of CFS function are not mutually exclusive but rather coexist; when breaks at CFSs are not repaired accurately, this can lead to deletions by which cells acquire growth advantage because of loss of tumor suppressor activities. Here, we review recent advances linking some CFS gene products with the DDR, genomic instability, and carcinogenesis and discuss how their inactivation might represent a selective advantage for cancer cells. PMID:27977694

  2. Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis

    PubMed Central

    Pedroza-Garcia, José Antonio; Domenichini, Séverine; Mazubert, Christelle; Bourge, Mickael; White, Charles; Hudik, Elodie; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; del Olmo, Ivan; Piñeiro, Manuel; Jarillo, Jose Antonio; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2016-01-01

    Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types. PMID:27193996

  3. DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.

    PubMed

    Bierle, Lindsey A; Reich, Kira L; Taylor, Braden E; Blatt, Eliot B; Middleton, Sydney M; Burke, Shawnecca D; Stultz, Laura K; Hanson, Pamela K; Partridge, Janet F; Miller, Mary E

    2015-01-01

    Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage--including that induced by many anticancer drugs--results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events.

  4. DNA Damage and Pulmonary Hypertension

    PubMed Central

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  5. Nesprin-2-dependent ERK1/2 compartmentalisation regulates the DNA damage response in vascular smooth muscle cell ageing

    PubMed Central

    Warren, D T; Tajsic, T; Porter, L J; Minaisah, R M; Cobb, A; Jacob, A; Rajgor, D; Zhang, Q P; Shanahan, C M

    2015-01-01

    Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs. PMID:25744025

  6. Identification of Cell Nonautonomous DNA Damage Responses in the Tumor Microenvironment that Contribute to Cancer Therapy Resistance

    DTIC Science & Technology

    2014-05-01

    include susceptibilities to toxic insults. Using a genome-wide analysis of transcriptional responses to genotoxic stress induced by cancer therapeutics...enhance resistance to DNA-damaging cancer therapeutics. These results suggest a mechanism by which genotoxic therapies given in a cyclical fashion...responses to genotoxic stress induced by cancer therapeutics, we have identified a spectrum of secreted proteins derived from the tumor microenvironment

  7. The role of the cancer stem cell marker CD271 in DNA damage response and drug resistance of melanoma cells

    PubMed Central

    Redmer, T; Walz, I; Klinger, B; Khouja, S; Welte, Y; Schäfer, R; Regenbrecht, C

    2017-01-01

    Several lines of evidence have suggested that stemness and acquired resistance to targeted inhibitors or chemotherapeutics are mechanistically linked. Here we observed high cell surface and total levels of nerve growth factor receptor/CD271, a marker of melanoma-initiating cells, in sub-populations of chemoresistant cell lines. CD271 expression was increased in drug-sensitive cells but not resistant cells in response to DNA-damaging chemotherapeutics etoposide, fotemustine and cisplatin. Comparative analysis of melanoma cells engineered to stably express CD271 or a targeting short hairpin RNA by expression profiling provided numerous genes regulated in a CD271-dependent manner. In-depth analysis of CD271-responsive genes uncovered the association of CD271 with regulation of DNA repair components. In addition, gene set enrichment analysis revealed enrichment of CD271-responsive genes in drug-resistant cells, among them DNA repair components. Moreover, our comparative screen identified the fibroblast growth factor 13 (FGF13) as a target of CD271, highly expressed in chemoresistant cells. Further we show that levels of CD271 determine drug response. Knock-down of CD271 in fotemustine-resistant cells decreased expression of FGF13 and at least partly restored sensitivity to fotemustine. Together, we demonstrate that expression of CD271 is responsible for genes associated with DNA repair and drug response. Further, we identified 110 CD271-responsive genes predominantly expressed in melanoma metastases, among them were NEK2, TOP2A and RAD51AP1 as potential drivers of melanoma metastasis. In addition, we provide mechanistic insight in the regulation of CD271 in response to drugs. We found that CD271 is potentially regulated by p53 and in turn is needed for a proper p53-dependent response to DNA-damaging drugs. In summary, we provide for the first time insight in a CD271-associated signaling network connecting CD271 with DNA repair, drug response and metastasis. PMID

  8. The C. elegans gene pme-5: molecular cloning and role in the DNA-damage response of a tankyrase orthologue.

    PubMed

    Gravel, Catherine; Stergiou, Lilli; Gagnon, Steve N; Desnoyers, Serge

    2004-02-03

    Tankyrases are recently identified proteins characterized by ankyrin repeats and a poly(ADP-ribose) polymerase (PARP) signature motif. In vertebrates, tankyrases mediate protein-protein interactions via the ankyrin domain. Many partners have been identified that could function in telomere maintenance, signal transduction in vesicular transport, and cell death. To further our knowledge of tankyrases and to study their function in development, we sought and found a tankyrase-related gene in Caenorhabditis elegans that we named pme-5 (poly(ADP-ribose) metabolism enzyme-5). The protein encoded includes a large ankyrin domain and a catalytic PARP domain containing the well-conserved PARP signature sequence and the regulatory region. Unlike other tankyrases, PME-5 lacks a sterile-alpha module (SAM), but has a coiled coil domain which may mediate oligomerization. We also found that pme-5 mRNA is alternatively spliced at the fifth exon, producing a long (PME-5L) and a short (PME-5S) transcript. Both isoforms are constitutively expressed during the life cycle of C. elegans. We also show DNA damage increases expression of pme-5, a response that requires the DNA damage checkpoint gene hus-1. Moreover, DNA damage-induced germ cell apoptosis was slightly increased in pme-5(RNAi) hermaphrodites. Altogether, these data indicate that pme-5 is part of a DNA damage response pathway which leads to apoptosis in C. elegans.

  9. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

    PubMed

    Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-01-01

    Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

  10. Regulation of the life cycle of HPVs by differentiation and the DNA damage response

    PubMed Central

    Hong, Shiyuan; Laimins, Laimonis A.

    2014-01-01

    HPVs are the causative agents of cervical and other anogenital cancers. HPVs infect stratified epithelia and link their productive life cycles to cellular differentiation. Low levels of viral genomes are stably maintained in undifferentiated cells and productive replication or amplification is restricted to differentiated suprabasal cells. Amplification is dependent on the activation of the ATM DNA damage factors that are recruited to viral replication centers and inhibition of this pathway blocks productive replication. The STAT-5 protein appears to play a critical role in mediating activation of the ATM pathway in HPV-positive cells. While HPVs need to activate the DNA damage pathway for replication, cervical cancers contain many genomic alterations suggesting that this pathway is circumvented during progression to malignancy. PMID:24266355

  11. Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells

    PubMed Central

    Cagnetta, Antonia; Adamia, Sophia; Acharya, Chirag; Tai, Yu-Tzu; Fulciniti, Mariateresa; Ohguchi, Hiroto; Munshi, Aditya; Acharya, Prakrati; Bhasin, Manoj K.; Zhong, Lei; Carrasco, Ruben; Monacelli, Fiammetta; Ballestrero, Alberto; Richardson, Paul; Gobbi, Marco; Lemoli, Roberto M.; Munshi, Nikhil; Hideshima, Teru; Nencioni, Alessio; Chauhan, Dharminder; Anderson, Kenneth C.

    2016-01-01

    Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD+-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM. PMID:26675349

  12. Impact of the Circadian Clock on UV-Induced DNA Damage Response and Photocarcinogenesis.

    PubMed

    Dakup, Panshak; Gaddameedhi, Shobhan

    2017-01-01

    The skin is in constant exposure to various external environmental stressors, including solar ultraviolet (UV) radiation. Various wavelengths of UV light are absorbed by the DNA and other molecules in the skin to cause DNA damage and induce oxidative stress. The exposure to excessive ultraviolet (UV) radiation and/or accumulation of damage over time can lead to photocarcinogenesis and photoaging. The nucleotide excision repair (NER) system is the sole mechanism for removing UV photoproduct damage from DNA, and genetic disruption of this repair pathway leads to the photosensitive disorder xeroderma pigmentosum (XP). Interestingly, recent work has shown that NER is controlled by the circadian clock, the body's natural time-keeping mechanism, through regulation of the rate-limiting repair factor xeroderma pigmentosum group A (XPA). Studies have shown reduced UV-induced skin cancer after UV exposure in the evening compared to the morning, which corresponds with times of high and low repair capacities, respectively. However, most studies of the circadian clock-NER connection have utilized murine models, and it is therefore important to translate these findings to humans to improve skin cancer prevention and chronotherapy.

  13. Suppression of the DNA damage response in acute myeloid leukemia versus myelodysplastic syndrome.

    PubMed

    Boehrer, S; Adès, L; Tajeddine, N; Hofmann, W K; Kriener, S; Bug, G; Ottmann, O G; Ruthardt, M; Galluzzi, L; Fouassier, C; Tailler, M; Olaussen, K A; Gardin, C; Eclache, V; de Botton, S; Thepot, S; Fenaux, P; Kroemer, G

    2009-06-04

    The molecular mechanisms responsible for the evolution from the preleukemic entities of low-risk myelodysplastic syndrome (MDS) to the less favorable forms of high-risk MDS, as well as those enabling transformation to acute myeloid leukemia (AML), are still incompletely understood. Abundant evidence from solid tumors demonstrates that preneoplastic lesions activate signaling pathways of a DNA damage response (DDR), which functions as an 'anticancer barrier' hindering tumorigenesis. Testing the hypothesis that subgroups of MDS and AML differ with respect to DDR, we first assessed markers of DDR (phosphorylation of ATM, Chk-1, Chk-2 and H2AX) in cell lines representing different entities of MDS (P39, MOLM-13) and AML (MV4-11, KG-1) before and after gamma-irradiation. Although gamma-irradiation induced apoptosis and G(2)/M arrest and a concomitant increase in the phosphorylation of ATM, Chk-1 and H2AX in MDS-derived cell lines, this radiation response was attenuated in the AML-derived cell lines. It is noteworthy that KG-1, but not P39 cells exhibit signs of an endogenous activation of the DDR. Similarly, we found that the frequency of P-ATM(+) cells detectable in bone marrow (BM) biopsies increased in samples from patients with AML as compared with high-risk MDS samples and significantly correlated with the percentage of BM blasts. In contrast, the frequency of gamma-H2AX(+) cells was heterogeneous in all subgroups of AML and MDS. Whereas intermediate-1 MDS samples contained as little P-Chk-1 and P-Chk-2 as healthy controls, staining for both checkpoint kinases increased in intermediate-2 and high-risk MDS, yet declined to near-to-background levels in AML samples. Thus the activation of Chk-1 and Chk-2 behaves in accord with the paradigm established for solid tumors, whereas ATM is activated during and beyond transformation. In conclusion, we demonstrate the heterogeneity of the DDR response in MDS and AML and provide evidence for its selective suppression in AML

  14. Cellular responses and gene expression profile changes due to bleomycin-induced DNA damage in human fibroblasts in space

    PubMed Central

    Kidane, Yared; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

    2017-01-01

    Living organisms in space are constantly exposed to radiation, toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage is essential for assessing the radiation risk for astronauts and the mutation rate in microorganisms. In a study conducted on the International Space Station, confluent human fibroblasts in culture were treated with bleomycin for three hours in the true microgravity environment. The degree of DNA damage was quantified by immunofluorescence staining for γ-H2AX, which is manifested in three types of staining patterns. Although similar percentages of these types of patterns were found between flight and ground cells, there was a slight shift in the distribution of foci counts in the flown cells with countable numbers of γ-H2AX foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. We also performed a microarray analysis of gene expressions in response to bleomycin treatment. A qualitative comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. The microarray data was confirmed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly upregulated in both flight and ground cells after bleomycin treatment. Our results suggest that whether microgravity affects DNA damage response in space can be dependent on the cell type and cell growth condition. PMID:28248986

  15. DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae

    PubMed Central

    Bierle, Lindsey A.; Reich, Kira L.; Taylor, Braden E.; Blatt, Eliot B.; Middleton, Sydney M.; Burke, Shawnecca D.; Stultz, Laura K.; Hanson, Pamela K.; Partridge, Janet F.; Miller, Mary E.

    2015-01-01

    Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage–including that induced by many anticancer drugs–results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events. PMID:26375390

  16. Promoter elements of the PHR1 gene of Saccharomyces cerevisiae and their roles in the response to DNA damage.

    PubMed Central

    Sancar, G B; Ferris, R; Smith, F W; Vandeberg, B

    1995-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the apoenzyme for the DNA repair enzyme photolyase. PHR1 transcription is induced in response to 254 nm radiation and a variety of chemical damaging agents. We report here the identification of promoter elements required for PHR1 expression. Transcription is regulated primarily through three sequence elements clustered within a 120 bp region immediately upstream of the translational start site. A 20 bp interrupted palindrome comprises UASPHR1 and is responsible for 80-90% of basal and induced expression. UASPHR1 alone can activate transcription of a CYC1 minimal promoter but does not confer damage responsiveness. In the intact PHR1 promoter UAS function is dependent upon an upstream essential sequence (UES). URSPHR1 contains a binding site for the damage-responsive repressor Prp; consistent with this role, deletion or specific mutations of the URS increase basal level expression and decrease the induction ratio. Deletion of URSPHR1 also eliminates the requirement for UESPHR1 for promoter activation, indicating that the UES attenuates Prp-mediated repression. Sequences within UASPHR1 are similar to regulatory sequences found upstream of both damage responsive and nonresponsive genes involved in DNA repair and metabolism. PMID:7501452

  17. Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage.

    PubMed Central

    Goerlich, O; Quillardet, P; Hofnung, M

    1989-01-01

    The induction of the SOS response by H2O2 was measured in Escherichia coli by means of a sfiA::lacZ operon fusion. The effects of mutations in genes involved in DNA repair or DNA metabolism on the SOS response were investigated. We found that in an uvrA mutant, H2O2 induced the SOS response at lower concentrations than in the uvr+ parent strain, indicating that some lesions induced by H2O2 may be repaired by the uvrABC-dependent excision repair system. A nth mutation, yielding deficiency in thymine glycol DNA glycosylase, had no detectable effect on SOS induction, indicating that thymine glycol, a DNA lesion expected to be induced by H2O2, does not participate detectably in the induction of the SOS response by this chemical under our conditions. H2O2 still induced the SOS response in a dnaC(Ts) uvrA double mutant under conditions in which no DNA replication proceeds, suggesting that this chemical induces DNA strand breaks. Induction of the SOS response by H2O2 was also assayed in various mutants affected in genes suspected to be important for protection against oxidative stress. Mutations in the catalase genes, katE and katG, had only minor effects. However, in an oxyR deletion mutant, in which the adaptative response to H2O2 does not occur, SOS induction occurred at much lower H2O2 concentrations than in the oxyR+ parent strain. These results indicate that some enzymes regulated by the oxyR gene are, under our conditions, more important than catalase for protection against the H2O2-induced DNA damages which trigger the SOS response. PMID:2681154

  18. Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

    SciTech Connect

    Jarvis, Ian W.H.; Bergvall, Christoffer; Bottai, Matteo; Westerholm, Roger; Stenius, Ulla; Dreij, Kristian

    2013-02-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower BP equivalent concentrations in air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAH-contaminated soil (Niziolek-Kierecka et al., 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHs. -- Highlights: ► Benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP) and air PM PAH extracts were compared. ► Binary mixture of BP and DBP induced a more than additive DNA damage response. ► Air PM PAH extracts were more potent than toxicity equivalency factor estimates. ► Larger PAHs (> 4 rings) contribute more to the genotoxicity of PAHs in air PM. ► Chk1 is a sensitive marker for persistent activation of DNA damage signaling from PAH mixtures.

  19. Cytogenetic responses to ionizing radiation exposure of human fibroblasts with knocked-down expressions of various DNA damage signaling genes

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Rohde, Larry; Wu, Honglu

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with up-regulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. Here, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yields of MN and/or CA formation were significantly increased by suppressed expression of some of the selected genes in DSB and other DNA repair pathways. Knocked-down expression of other genes showed significant impact on cell cycle progression, possibly because of severe impairment of DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  20. DNA damage and carcinogenesis

    SciTech Connect

    Stelow, R B

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10/sup 4/ fold.

  1. Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling

    PubMed Central

    Chaki, Moumita; Airik, Rannar; Ghosh, Amiya K.; Giles, Rachel H.; Chen, Rui; Slaats, Gisela G.; Wang, Hui; Hurd, Toby W.; Zhou, Weibin; Cluckey, Andrew; Gee, Heon-Yung; Ramaswami, Gokul; Hong, Chen-Jei; Hamilton, Bruce A.; Červenka, Igor; Ganji, Ranjani Sri; Bryja, Vitezslav; Arts, Heleen H.; van Reeuwijk, Jeroen; Oud, Machteld M.; Letteboer, Stef J.F.; Roepman, Ronald; Husson, Hervé; Ibraghimov-Beskrovnaya, Oxana; Ysunaga, Takayuki; Walz, Gerd; Eley, Lorraine; Sayer, John A.; Schermer, Bernhard; Liebau, Max C.; Benzing, Thomas; Le Corre, Stephanie; Drummond, Iain; Joles, Jaap A.; Janssen, Sabine; Allen, Susan J.; Natarajan, Sivakumar; O Toole, John F.; Attanasio, Massimo; Saunier, Sophie; Antignac, Corinne; Koenekoop, Robert K.; Ren, Huanan; Lopez, Irma; Nayir, Ahmet; Stoetzel, Corinne; Dollfus, Helene; Massoudi, Rustin; Gleeson, Joseph G.; Andreoli, Sharon P.; Doherty, Dan G.; Lindstrad, Anna; Golzio, Christelle; Katsanis, Nicholas; Pape, Lars; Abboud, Emad B.; Al-Rajhi, Ali A.; Lewis, Richard A.; Lupski, James R.; Omran, Heymut; Lee, Eva; Wang, Shaohui; Sekiguchi, JoAnn M.; Saunders, Rudel; Johnson, Colin A.; Garner, Elizabeth; Vanselow, Katja; Andersen, Jens S.; Shlomai, Joseph; Nurnberg, Gudrun; Nurnberg, Peter; Levy, Shawn; Smogorzewska, Agata; Otto, Edgar A.; Hildebrandt, Friedhelm

    2012-01-01

    SUMMARY Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as ‘ciliopathies’. However, disease mechanisms remain poorly understood. Here we identify by whole exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway, hitherto not implicated in ciliopathies. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164 and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents, and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR. PMID:22863007

  2. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins.

    PubMed

    Lisby, Michael; Barlow, Jacqueline H; Burgess, Rebecca C; Rothstein, Rodney

    2004-09-17

    DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

  3. Evidence for hSNM1B/Apollo functioning in the HSP70 mediated DNA damage response.

    PubMed

    Anders, Marco; Mattow, Jens; Digweed, Martin; Demuth, Ilja

    2009-06-01

    The hSNM1B/Apollo protein is involved in the cellular response to DNA-damage as well as in the maintenance of telomeres during S-phase. TRF2 has been shown to interact physically with hSNM1B. As a core component of shelterin, TRF2 functions in organization and protection of telomeres. However, TRF2 was also shown to have a role in the early DNA-damage response, suggesting that hSNM1B and TRF2 cooperate in this dual function. Here we have used Tandem-Affinity-Purification in combination with mass spectrometry to identify additional binding partners of hSNM1B. This revealed HSC70, HSP72, HSP60 and beta-Tubulin to be hSNM1B-interactors. We have confirmed the interaction of hSNM1B and HSP70 in co-immunoprecipitation assays and found that hSNM1B binds to a C-terminal fragment of HSP72, known to contain the substrate binding domain. Depletion of HSP72 in human fibroblasts resulted in a significant reduction of nuclear hSNM1B foci. We also found the phosphorylation of CHK1 at serine 317 to be attenuated in response to UVC irradiation as a consequence of hSNM1B depletion, a result which extends our previous findings on the DNA-damage response function of hSNM1B. HSP70 chaperones have been implicated in the maintenance of genome stability and their expression is often aberrant in cancer. Our results presented here, suggest that the role in genome stability might not be specific to HSP70 but rather can be attributed, at least in part, to hSNM1B. This, together with its stimulating effect on ATM and ATR substrate phosphorylation in response to DNA-damage qualify hSNM1B as a putative target in cancer therapy.

  4. A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses

    SciTech Connect

    Preta, Giulio; Klark, Rainier de; Glas, Rickard

    2009-11-27

    Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to {gamma}-irradiation, and that nuclear expression of TPPII was present in most {gamma}-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after {gamma}-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following {gamma}-irradiation (at 1-4 h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in {gamma}-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

  5. Protein kinase C{eta} activates NF-{kappa}B in response to camptothecin-induced DNA damage

    SciTech Connect

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

    2011-08-26

    Highlights: {yields} Protein kinase C-eta (PKC{eta}) is an upstream regulator of the NF-{kappa}B signaling pathway. {yields} PKC{eta} activates NF-{kappa}B in non-stressed conditions and in response to DNA damage. {yields} PKC{eta} regulates NF-{kappa}B by activating I{kappa}B kinase (IKK) and inducing I{kappa}B degradation. -- Abstract: The nuclear factor {kappa}B (NF-{kappa}B) 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-{kappa}B in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-{kappa}B regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKC{eta}) regulates NF-{kappa}B upstream signaling by activating the I{kappa}B kinase (IKK) and the degradation of I{kappa}B. Furthermore, PKC{eta} enhances the nuclear translocation and transactivation of NF-{kappa}B under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKC{eta} confers protection against DNA damage-induced apoptosis. Our present study suggests that PKC{eta} is involved in NF-{kappa}B signaling leading to drug resistance.

  6. Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.

    PubMed

    Bernas, Tytus; Berniak, Krzysztof; Rybak, Paulina; Zarębski, Mirosław; Zhao, Hong; Darzynkiewicz, Zbigniew; Dobrucki, Jerzy W

    2013-10-01

    Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns. Such a correlation can be used to estimate a probability of inducing damage in sections of replicating DNA. The damage and replication foci are imaged in 3D with confocal microscopy and their respective positions within nuclei are determined with adaptive image segmentation. Using correlation functions spatial proximity of the resultant point patterns is quantified over the range of distances in cells in early-, mid- and late S-phase. As the numbers (and nuclear densities) of γH2AX and replication foci differ significantly in the subsequent substages of S phase, the detected association values were corrected for the expected random overlap between both classes of foci. Thus, the probability of their nonrandom association was estimated. Moreover, self association (clustering) of DNA replication sites in different stages of S-phase of the cell cycle was detected and accounted for. While the analysis revealed a strong correlation between the γH2AX foci and the sites of DNA replication in cells treated with Cpt, only a low correlation was apparent in cells exposed to H2O2. © 2013 International Society for Advancement of Cytometry.

  7. The Deubiquitinase USP9X Maintains DNA Replication Fork Stability and DNA Damage Checkpoint Responses by Regulating CLASPIN during S-Phase.

    PubMed

    McGarry, Edel; Gaboriau, David; Rainey, Michael D; Restuccia, Umberto; Bachi, Angela; Santocanale, Corrado

    2016-04-15

    Coordination of the multiple processes underlying DNA replication is key for maintaining genome stability and preventing tumorigenesis. CLASPIN, a critical player in replication fork stabilization and checkpoint responses, must be tightly regulated during the cell cycle to prevent the accumulation of DNA damage. In this study, we used a quantitative proteomics approach and identified USP9X as a novel CLASPIN-interacting protein. USP9X is a deubiquitinase involved in multiple signaling and survival pathways whose tumor suppressor or oncogenic activity is highly context dependent. We found that USP9X regulated the expression and stability of CLASPIN in an S-phase-specific manner. USP9X depletion profoundly impairs the progression of DNA replication forks, causing unscheduled termination events with a frequency similar to CLASPIN depletion, resulting in excessive endogenous DNA damage. Importantly, restoration of CLASPIN expression in USP9X-depleted cells partially suppressed the accumulation of DNA damage. Furthermore, USP9X depletion compromised CHK1 activation in response to hydroxyurea and UV, thus promoting hypersensitivity to drug-induced replication stress. Taken together, our results reveal a novel role for USP9X in the maintenance of genomic stability during DNA replication and provide potential mechanistic insights into its tumor suppressor role in certain malignancies. Cancer Res; 76(8); 2384-93. ©2016 AACR.

  8. Diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication.

    PubMed

    Marriott, Andrew S; Copeland, Nikki A; Cunningham, Ryan; Wilkinson, Mark C; McLennan, Alexander G; Jones, Nigel J

    2015-09-01

    The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 μM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA.

  9. G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells

    PubMed Central

    Hasegawa, Daiki; Okabe, Sachiko; Okamoto, Keiji; Nakano, Ichiro; Shin-ya, Kazuo; Seimiya, Hiroyuki

    2016-01-01

    Glioblastoma (GBM) is an invariably fatal brain tumor in which a small subpopulation of self-renewable glioma stem cells (GSCs) contributes to tumor propagation and relapse. Targeting GSCs could therefore have a significant clinical impact for GBM. Telomestatin is a naturally-occurring compound that preferentially impairs GSC growth by perturbing transcription and inducing a DNA damage response. Telomestatin stabilizes G-quadruplexes (G4s), which are guanine-rich four-strand nucleic acid structures observed in vitro and in vivo. However, the mechanism underlying the GSC-selective nature of the DNA damage response remains unknown. Here we demonstrate that GSCs are more susceptible to telomestatin-induced telomere dysfunction and replication stress when compared with GSC-derived non-stem glioma cells (NSGCs). Telomestatin induced dissociation of the telomere-capping protein TRF2 from telomeres, leading to telomeric DNA damage in GSCs–but not in NSGCs. BIBR1532, a telomerase catalytic inhibitor, did not preferentially inhibit GSC growth, suggesting that telomestatin promotes telomere dysfunction in a telomerase-independent manner. GSCs and NSGCs had comparable levels of G4s in their nuclei, and both responded to telomestatin with phosphorylation of RPA2 at Ser33–a hallmark of replication stress. However, activation of the checkpoint kinase Chk1, induction of a DNA damage response, and subsequent growth inhibition occurred only in telomestatin-treated GSCs. These observations suggest that telomestatin impairs GSC growth through removal of TRF2 from telomeres and potent activation of the replication stress response pathway. Therefore, a novel G4-directed therapeutic strategy could specifically target cancer stem cells in GBM. PMID:26845351

  10. Selection and the cell cycle: positive Darwinian selection in a well-known DNA damage response pathway.

    PubMed

    O'Connell, Mary J

    2010-12-01

    Cancer is a common occurrence in multi-cellular organisms and is not strictly limited to the elderly in a population. It is therefore possible that individuals with genotypes that protect against early onset cancers have a selective advantage. In this study the patterns of mutation in the proteins of a well-studied DNA damage response pathway have been examined for evidence of adaptive evolutionary change. Using a maximum likelihood framework and the mammalian species phylogeny, together with codon models of evolution, selective pressure variation across the interacting network of proteins has been detected. The presence of signatures of adaptive evolution in BRCA1 and BRCA2 has already been documented but the effect on the entire network of interacting proteins in this damage response pathway has, until now, been unknown. Positive selection is evident throughout the network with a total of 11 proteins out of 15 examined displaying patterns of substitution characteristic of positive selection. It is also shown here that modern human populations display evidence of an ongoing selective sweep in 9 of these DNA damage repair proteins. The results presented here provide the community with new residues that may be relevant to cancer susceptibility while also highlighting those proteins where human and mouse have undergone lineage-specific functional shift. An understanding of this damage response pathway from an evolutionary perspective will undoubtedly contribute to future cancer treatment approaches.

  11. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E2, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional “repair and survive, or die” hypothesis. PMID:27187358

  12. A conserved DNA damage response pathway responsible for coupling the cell division cycle to the circadian and metabolic cycles.

    PubMed

    Chen, Zheng; McKnight, Steven L

    2007-12-01

    The circadian clock drives endogenous oscillations of cellular and physiological processes with a periodicity of approximately 24 h. Progression of the cell division cycle (CDC) has been found to be coupled to the circadian clock, and it has been postulated that gating of the CDC by the circadian cycle may have evolved to protect DNA from the mutagenic effects of ultraviolet light. When grown under nutrient-limiting conditions in a chemostat, prototrophic strains of budding yeast, Saccharomyces cerevisiae, adopt a robust metabolic cycle of ultradian dimensions that temporally compartmentalizes essential cellular events. The CDC is gated by this yeast metabolic cycle (YMC), with DNA replication strictly segregated away from the oxidative phase when cells are actively respiring. Mutants impaired in such gating allow DNA replication to take place during the respiratory phase of the YMC and have been found to suffer significantly elevated rates of spontaneous mutation. Analogous to the circadian cycle, the YMC also employs the conserved DNA checkpoint kinase Rad53/Chk2 to facilitate coupling with the CDC. These studies highlight an evolutionarily conserved mechanism that seems to confine cell division to particular temporal windows to prevent DNA damage. We hypothesize that DNA damage itself might constitute a "zeitgeber", or time giver, for both the circadian cycle and the metabolic cycle. We discuss these findings in the context of a unifying theme underlying the circadian and metabolic cycles, and explore the relevance of cell cycle gating to human diseases including cancer.

  13. The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.

    PubMed

    Takada, Saeko; Collins, Eric R; Kurahashi, Kayo

    2015-05-15

    DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.

  14. The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses

    PubMed Central

    Takada, Saeko; Collins, Eric R.; Kurahashi, Kayo

    2015-01-01

    DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage. PMID:25808488

  15. 17β estradiol induced ROS generation, DNA damage and enzymatic responses in the hepatic tissue of Japanese sea bass.

    PubMed

    Thilagam, Harikrishnan; Gopalakrishnan, Singaram; Qu, Hai-Dong; Bo, Jun; Wang, Ke-Jian

    2010-10-01

    The importance of endocrine disrupting chemicals and their effects on fish has been documented in recent years. However, little is known about whether the estrogenic compound 17β estradiol (E2) causes oxidative stress in the hepatic tissue of fish. Therefore, this work tested the hypothesis that E2 might cause oxidative stress in the Japanese sea bass Lateolabrax japonicus liver. To test this hypothesis, its effects on reactive oxygen species (ROS) production, DNA damage, antioxidants and biotransformation enzyme were investigated in two different size groups (fingerling and juvenile groups) following 30 days exposure. Results showed that there was a good relationship between the E2 exposure concentration, plasma E2 level and ROS generation. In addition ROS production correlated negatively with 7-ethoxyresorufin-O-deethylase activity and positively with DNA damage and lipid peroxidation (LPO). Antioxidant enzymes such as superoxide dismutase and catalase did not show any significant relation with ROS, LPO and DNA damage. In contrast, glutathione mediated enzymes showed a good relationship with the above parameters suggesting that the glutathione system in fish might be responsible for protection against the impact of E2 and also indicating a possible adaptive response during exposure periods. In addition, it was observed that fingerling was more susceptible to E2 exposure than juvenile fish. The present study provided strong evidence that the ROS level increased significantly in the liver of E2 exposed fish, and that ROS might serve as a biomarker to indicate estrogen contamination.

  16. Compartmental stress responses correlate with cell survival in bystander effects induced by the DNA damage agent, bleomycin.

    PubMed

    Savu, Diana; Petcu, Ileana; Temelie, Mihaela; Mustaciosu, Cosmin; Moisoi, Nicoleta

    2015-01-01

    Physical or chemical stress applied to a cell system trigger a signal cascade that is transmitted to the neighboring cell population in a process known as bystander effect. Despite its wide occurrence in biological systems this phenomenon is mainly documented in cancer treatments. Thus understanding whether the bystander effect acts as an adaptive priming element for the neighboring cells or a sensitization factor is critical in designing treatment strategies. Here we characterize the bystander effects induced by bleomycin, a DNA-damaging agent, and compartmental stress responses associated with this phenomenon. Mouse fibroblasts were treated with increasing concentrations of bleomycin and assessed for DNA damage, cell death and induction of compartmental stress response (endoplasmic reticulum, mitochondrial and cytoplasmic stress). Preconditioned media were used to analyze bystander damage using the same end-points. Bleomycin induced bystander response was reflected primarily in increased DNA damage. This was dependent on the concentration of bleomycin and time of media conditioning. Interestingly, we found that ROS but not NO are involved in the transmission of the bystander effect. Consistent transcriptional down-regulation of the stress response factors tested (i.e. BiP, mtHsp60, Hsp70) occurred in the direct effect indicating that bleomycin might induce an arrest of transcription correlated with decreased survival. We observed the opposite trend in the bystander effect, with specific stress markers appearing increased and correlated with increased survival. These data shed new light on the potential role of stress pathways activation in bystander effects and their putative impact on the pro-survival pro-death balance.

  17. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells.

    PubMed

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian; Thomale, Jürgen; Schupp, Nicole; Fritz, Gerhard

    2016-02-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury.

  18. Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response

    PubMed Central

    Ben Yehuda, Adi; Risheq, Marwa; Novoplansky, Ofra; Bersuker, Kirill; Kopito, Ron R.; Goldberg, Michal; Brandeis, Michael

    2017-01-01

    Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington’s disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality. PMID:28052107

  19. Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis.

    PubMed

    Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

    2017-03-09

    Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways.

  20. Kinetic Modeling Reveals the Roles of Reactive Oxygen Species Scavenging and DNA Repair Processes in Shaping the Dose-Response Curve of KBrO₃-Induced DNA Damage.

    PubMed

    Spassova, Maria A; Miller, David J; Nikolov, Alexander S

    2015-01-01

    We have developed a kinetic model to investigate how DNA repair processes and scavengers of reactive oxygen species (ROS) can affect the dose-response shape of prooxidant induced DNA damage. We used as an example chemical KBrO3 which is activated by glutathione and forms reactive intermediates that directly interact with DNA to form 8-hydroxy-2-deoxyguanosine DNA adducts (8-OH-dG). The single strand breaks (SSB) that can result from failed base excision repair of these adducts were considered as an effect downstream from 8-OH-dG. We previously demonstrated that, in the presence of effective base excision repair, 8-OH-dG can exhibit threshold-like dose-response dependence, while the downstream SSB can still exhibit a linear dose-response. Here we demonstrate that this result holds for a variety of conditions, including low levels of GSH, the presence of additional SSB repair mechanisms, or a scavenger. It has been shown that melatonin, a terminal scavenger, inhibits KBrO3-caused oxidative damage. Our modeling revealed that sustained exposure to KBrO3 can lead to fast scavenger exhaustion, in which case the dose-response shapes for both endpoints are not substantially affected. The results are important to consider when forming conclusions on a chemical's toxicity dose dependence based on the dose-response of early genotoxic events.

  1. Inner nuclear envelope proteins SUN1 and SUN2 play a prominent role in the DNA damage response

    PubMed Central

    Lei, Kai; Zhu, Xiaoqiang; Xu, Rener; Xu, Tian; Zhuang, Yuan; Han, Min

    2012-01-01

    Summary The DNA damage response (DDR) and DNA repair are critical for maintaining genomic stability and evading many human diseases [1, 2]. Recent findings indicate accumulation of SUN1, a nuclear envelope (NE) protein, is a significant pathogenic event in Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome, both caused by mutations in LMNA [3, 4]. However, roles of mammalian SUN proteins in mitotic cell division and genomic stability are unknown. Here we report that the inner NE proteins SUN1 and SUN2 may play a redundant role in DDR. Mouse embryonic fibroblasts from Sun1−/−Sun2−/− mice displayed premature proliferation arrest in S phase of cell cycle, increased apoptosis and DNA damage, and decreased perinuclear heterochromatin, indicating genome instability. Furthermore, activation of ATM and H2A.X, early events in DDR, were impaired in Sun1−/−Sun2−/− fibroblasts. A biochemical screen identified interactions between SUN1/2 and DNA-dependent protein kinase (DNAPK) complex that functions in DNA nonhomologous end joining repair and possibly in DDR [2, 5, 6]. Knockdown of DNAPK reduced ATM activation in NIH3T3 cells, consistent with a potential role of SUN1/2-DNAPK interaction during DDR. SUN1/2 could affect DDR by localizing certain nuclear factors to the NE or by mediating the communication between nuclear and cytoplasmic events. PMID:22863315

  2. Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses.

    PubMed

    Peng, Min; Xie, Jenny; Ucher, Anna; Stavnezer, Janet; Cantor, Sharon B

    2014-08-01

    Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.

  3. Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

    PubMed Central

    Lancini, Cesare; van den Berk, Paul C.M.; Vissers, Joseph H.A.; Gargiulo, Gaetano; Song, Ji-Ying; Hulsman, Danielle; Serresi, Michela; Tanger, Ellen; Blom, Marleen; Vens, Conchita; van Lohuizen, Maarten; Jacobs, Heinz

    2014-01-01

    Histone ubiquitination at DNA breaks is required for activation of the DNA damage response (DDR) and DNA repair. How the dynamic removal of this modification by deubiquitinating enzymes (DUBs) impacts genome maintenance in vivo is largely unknown. To address this question, we generated mice deficient for Ub-specific protease 3 (USP3; Usp3Δ/Δ), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling. Notably, USP3 deletion increased the levels of histone ubiquitination in adult tissues, reduced the hematopoietic stem cell (HSC) reserves over time, and shortened animal life span. Mechanistically, our data show that USP3 is important in HSC homeostasis, preserving HSC self-renewal, and repopulation potential in vivo and proliferation in vitro. A defective DDR and unresolved spontaneous DNA damage contribute to cell cycle restriction of Usp3Δ/Δ HSCs. Beyond the hematopoietic system, Usp3Δ/Δ animals spontaneously developed tumors, and primary Usp3Δ/Δ cells failed to preserve chromosomal integrity. These findings broadly support the regulation of chromatin ubiquitination as a key pathway in preserving tissue function through modulation of the response to genotoxic stress. PMID:25113974

  4. Humans and chimpanzees differ in their cellular response to DNA damage and non-coding sequence elements of DNA repair-associated genes.

    PubMed

    Weis, E; Galetzka, D; Herlyn, H; Schneider, E; Haaf, T

    2008-01-01

    Compared to humans, chimpanzees appear to be less susceptible to many types of cancer. Because DNA repair defects lead to accumulation of gene and chromosomal mutations, species differences in DNA repair are one plausible explanation. Here we analyzed the repair kinetics of human and chimpanzee cells after cisplatin treatment and irradiation. Dot blots for the quantification of single-stranded (ss) DNA repair intermediates revealed a biphasic response of human and chimpanzee lymphoblasts to cisplatin-induced damage. The early phase of DNA repair was identical in both species with a peak of ssDNA intermediates at 1 h after DNA damage induction. However, the late phase differed between species. Human cells showed a second peak of ssDNA intermediates at 6 h, chimpanzee cells at 5 h. One of four analyzed DNA repair-associated genes, UBE2A, was differentially expressed in human and chimpanzee cells at 5 h after cisplatin treatment. Immunofluorescent staining of gammaH2AX foci demonstrated equally high numbers of DNA strand breaks in human and chimpanzee cells at 30 min after irradiation and equally low numbers at 2 h. However, at 1 h chimpanzee cells had significantly less DNA breaks than human cells. Comparative sequence analyses of approximately 100 DNA repair-associated genes in human and chimpanzee revealed 13% and 32% genes, respectively, with evidence for an accelerated evolution in promoter regions and introns. This is strikingly contrasting to the 3% of DNA repair-associated genes with positive selection in the coding sequence. Compared to the rhesus macaque as an outgroup, chimpanzees have a higher accelerated evolution in non-coding sequences than humans. The TRF1-interacting, ankyrin-related ADP-ribose polymerase (TNKS) gene showed an accelerated intraspecific evolution among humans. Our results are consistent with the view that chimpanzee cells repair different types of DNA damage faster than human cells, whereas the overall repair capacity is similar in

  5. Oxidative DNA damage and inflammatory responses in cultured human cells and in humans exposed to traffic-related particles.

    PubMed

    Vattanasit, Udomratana; Navasumrit, Panida; Khadka, Man Bahadur; Kanitwithayanun, Jantamas; Promvijit, Jeerawan; Autrup, Herman; Ruchirawat, Mathuros

    2014-01-01

    Particulate pollution is a major public health concern because epidemiological studies have demonstrated that exposure to particles is associated with respiratory diseases and lung cancer. Diesel exhaust particles (DEP), which is classified as a human carcinogen (IARC, 2012), are considered a major contributor to traffic-related particulate matter (PM) in urban areas. DEP consists of various compounds, including PAHs and metals which are the principal components that contribute to the toxicity of PM. The present study aimed to investigate effects of PM on induction of oxidative DNA damage and inflammation by using lymphocytes in vitro and in human exposed to PM in the environment. Human lymphoblasts (RPMI 1788) were treated with DEP (SRM 2975) at various concentrations (25-100 μg/ml) to compare the extent of responses with alveolar epithelial cells (A549). ROS generation was determined in each cell cycle phase of DEP-treated cells in order to investigate the influence of the cell cycle stage on induction of oxidative stress. The oxidative DNA damage was determined by measurement of 8-hydroxy-deoxyguanosine (8-OHdG) whereas the inflammatory responses were determined by mRNA expression of interleukin-6 and -8 (IL-6 and IL-8), Clara cell protein (CC16), and lung surfactant protein-A (SP-A). The results showed that RPMI 1788 and A549 cells had a similar pattern of dose-dependent responses to DEP in terms of particle uptake, ROS generation with highest level found in G2/M phase, 8-OHdG formation, and induction of IL-6 and IL-8 expression. The human study was conducted in 51 healthy subjects residing in traffic-congested areas. The effects of exposure to PM2.5 and particle-bound PAHs and toxic metals on the levels of 8-OHdG in lymphocyte DNA, IL-8 expression in lymphocytes, and serum CC16 were evaluated. 8-OHdG levels correlated with the exposure levels of PM2.5 (P<0.01) and PAHs (P<0.05), but this was not the case with IL-8. Serum CC16 showed significantly negative

  6. Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response

    PubMed Central

    Edmonds, Matthew J.; Carter, Rachel J.; Nickson, Catherine M.; Williams, Sarah C.; Parsons, Jason L.

    2017-01-01

    Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo. We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response. PMID:27924031

  7. Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence ▿

    PubMed Central

    Aranda, Jesús; Bardina, Carlota; Beceiro, Alejandro; Rumbo, Soraya; Cabral, Maria P.; Barbé, Jordi; Bou, Germán

    2011-01-01

    RecA is the major enzyme involved in homologous recombination and plays a central role in SOS mutagenesis. In Acinetobacter spp., including Acinetobacter baumannii , a multidrug-resistant bacterium responsible for nosocomial infections worldwide, DNA repair responses differ in many ways from those of other bacterial species. In this work, the function of A. baumannii RecA was examined by constructing a recA mutant. Alteration of this single gene had a pleiotropic effect, showing the involvement of RecA in DNA damage repair and consequently in cellular protection against stresses induced by DNA damaging agents, several classes of antibiotics, and oxidative agents. In addition, the absence of RecA decreased survival in response to both heat shock and desiccation. Virulence assays in vitro (with macrophages) and in vivo (using a mouse model) similarly implicated RecA in the pathogenicity of A. baumannii . Thus, the data strongly suggest a protective role for RecA in the bacterium and indicate that inactivation of the protein can contribute to a combined therapeutic approach to controlling A. baumannii infections. PMID:21642465

  8. A novel role for non-ubiquitinated FANCD2 in response to hydroxyurea-induced DNA damage.

    PubMed

    Chen, X; Bosques, L; Sung, P; Kupfer, G M

    2016-01-07

    Fanconi anemia (FA) is a genetic disease of bone marrow failure, cancer susceptibility, and sensitivity to DNA crosslinking agents. FANCD2, the central protein of the FA pathway, is monoubiquitinated upon DNA damage, such as crosslinkers and replication blockers such as hydroxyurea (HU). Even though FA cells demonstrate unequivocal sensitivity to crosslinkers, such as mitomycin C (MMC), we find that they are largely resistant to HU, except for cells absent for expression of FANCD2. FANCD2, RAD51 and RAD18 form a complex, which is enhanced upon HU exposure. Surprisingly, although FANCD2 is required for this enhanced interaction, its monoubiquitination is not. Similarly, non-ubiquitinated FANCD2 can still support proliferation cell nuclear antigen (PCNA) monoubiquitination. RAD51, but not BRCA2, is also required for PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion (TLS) polymerase PolH chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knockdown cells and RAD51 siRNA knockdown cells, and PolH knockdown results in HU sensitivity only. Our data suggest that FANCD2 and RAD51 have an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with MMC treatment, suggesting a non-canonical function for the FA pathway in response to different types of DNA damage.

  9. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    PubMed Central

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J.; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M.; Bunting, Samuel F.; Inuzuka, Hiroyuki; Zheng, X. F. Steven; Shen, Zhiyuan; Sabaawy, Hatem E.; Liu, LianXin; Pine, Sharon R.

    2016-01-01

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance. PMID:27566146

  10. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage.

    PubMed

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M; Bunting, Samuel F; Inuzuka, Hiroyuki; Zheng, X F Steven; Shen, Zhiyuan; Sabaawy, Hatem E; Liu, LianXin; Pine, Sharon R

    2016-10-14

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance.

  11. The Response to Oxidative DNA Damage in Neurons: Mechanisms and Disease

    PubMed Central

    Narciso, Laura; Parlanti, Eleonora; Racaniello, Mauro; Simonelli, Valeria; Cardinale, Alessio; Merlo, Daniela; Dogliotti, Eugenia

    2016-01-01

    There is a growing body of evidence indicating that the mechanisms that control genome stability are of key importance in the development and function of the nervous system. The major threat for neurons is oxidative DNA damage, which is repaired by the base excision repair (BER) pathway. Functional mutations of enzymes that are involved in the processing of single-strand breaks (SSB) that are generated during BER have been causally associated with syndromes that present important neurological alterations and cognitive decline. In this review, the plasticity of BER during neurogenesis and the importance of an efficient BER for correct brain function will be specifically addressed paying particular attention to the brain region and neuron-selectivity in SSB repair-associated neurological syndromes and age-related neurodegenerative diseases. PMID:26942017

  12. Toxic Effect of Silica Nanoparticles on Endothelial Cells through DNA Damage Response via Chk1-Dependent G2/M Checkpoint

    PubMed Central

    Duan, Junchao; Yu, Yongbo; Li, Yang; Yu, Yang; Li, Yanbo; Zhou, Xianqing; Huang, Peili; Sun, Zhiwei

    2013-01-01

    Silica nanoparticles have become promising carriers for drug delivery or gene therapy. Endothelial cells could be directly exposed to silica nanoparticles by intravenous administration. However, the underlying toxic effect mechanisms of silica nanoparticles on endothelial cells are still poorly understood. In order to clarify the cytotoxicity of endothelial cells induced by silica nanoparticles and its mechanisms, cellular morphology, cell viability and lactate dehydrogenase (LDH) release were observed in human umbilical vein endothelial cells (HUVECs) as assessing cytotoxicity, resulted in a dose- and time- dependent manner. Silica nanoparticles-induced reactive oxygen species (ROS) generation caused oxidative damage followed by the production of malondialdehyde (MDA) as well as the inhibition of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Both necrosis and apoptosis were increased significantly after 24 h exposure. The mitochondrial membrane potential (MMP) decreased obviously in a dose-dependent manner. The degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM) were markedly aggravated. Silica nanoparticles also induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. In summary, our data indicated that the toxic effect mechanisms of silica nanoparticles on endothelial cells was through DNA damage response (DDR) via Chk1-dependent G2/M checkpoint signaling pathway, suggesting that exposure to silica nanoparticles could be a potential hazards for the development of cardiovascular diseases. PMID:23620807

  13. AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage

    PubMed Central

    Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

    2015-01-01

    Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3′ end of DNA damage-activated genes to facilitate transcriptional termination. PMID:25813038

  14. Phosphorylation of p53 by TAF1 inactivates p53-dependent transcription in the DNA damage response

    PubMed Central

    Wu, Yong; Lin, Joy C.; Piluso, Landon G.; Dhahbi, Joseph M.; Bobadilla, Selene; Spindler, Stephen R.; Liu, Xuan

    2014-01-01

    Summary While p53 activation has long been studied, the mechanisms by which its targets genes are restored to their pre-activation state are less clear. We report here that TAF1 phosphorylates p53 at Thr55, leading to dissociation of p53 from the p21 promoter and inactivation of transcription late in the DNA damage response. We further show that cellular ATP level might act as a molecular switch for Thr55 phosphorylation on the p21 promoter, indicating that TAF1 is a cellular ATP sensor. Upon DNA damage, cells undergo PARP-1-dependent ATP depletion, which is correlated with reduced TAF1 kinase activity and Thr55 phosphorylation, resulting in p21 activation. As cellular ATP levels recover, TAF1 is able to phosphorylate p53 on Thr55, which leads to dissociation of p53 from the p21 promoter. ChIP-sequencing analysis reveals p53 dissociates from promoters genome-wide as cells recover from DNA damage, suggesting the general nature of this mechanism. PMID:24289924

  15. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

    PubMed

    Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

    2015-01-01

    Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

  16. Suppression of Akt-mTOR Pathway-A Novel Component of Oncogene Induced DNA Damage Response Barrier in Breast Tumorigenesis

    PubMed Central

    Bhardwaj, Anjana; Rosen, Daniel; Liu, Mei; Liu, Yan; Hao, Qiang; Ganesan, Nivetha; Etzel, Carol J.; Gullett, Ashley; Albarracin, Constance T.; Bedrosian, Isabelle

    2014-01-01

    DNA damage has been thought to be directly associated with the neoplastic progression by enabling mutations in tumor suppressor genes and activating/and amplifying oncogenes ultimately resulting in genomic instability. DNA damage causes activation of the DNA damage response (DDR) that is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. In the present study, by using breast tissues samples from women at different risk levels for invasive breast cancer (normal, proliferative breast disease and ductal carcinoma in situ) we found that DNA damage is inversely correlated with risk of invasive breast cancer. Similarly, in MCF10A based in vitro model system where we recapitulated high DNA damage conditions as seen in patient samples by stably cloning in cyclin E, we found that high levels of oncogene induced DNA damage, by triggering inhibition of a major proliferative pathway (AKT), inhibits cell growth and causes cells to die through autophagy. These data suggest that AKT-mTOR pathway is a novel component of oncogene induced DNA damage response in immortalized ‘normal-like’ breast cells and its suppression may contribute to growth arrest and arrest of the breast tumorigenesis. PMID:24811059

  17. Suppression of Akt-mTOR pathway-a novel component of oncogene induced DNA damage response barrier in breast tumorigenesis.

    PubMed

    Bhardwaj, Anjana; Rosen, Daniel; Liu, Mei; Liu, Yan; Hao, Qiang; Ganesan, Nivetha; Etzel, Carol J; Gullett, Ashley; Albarracin, Constance T; Bedrosian, Isabelle

    2014-01-01

    DNA damage has been thought to be directly associated with the neoplastic progression by enabling mutations in tumor suppressor genes and activating/and amplifying oncogenes ultimately resulting in genomic instability. DNA damage causes activation of the DNA damage response (DDR) that is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. In the present study, by using breast tissues samples from women at different risk levels for invasive breast cancer (normal, proliferative breast disease and ductal carcinoma in situ) we found that DNA damage is inversely correlated with risk of invasive breast cancer. Similarly, in MCF10A based in vitro model system where we recapitulated high DNA damage conditions as seen in patient samples by stably cloning in cyclin E, we found that high levels of oncogene induced DNA damage, by triggering inhibition of a major proliferative pathway (AKT), inhibits cell growth and causes cells to die through autophagy. These data suggest that AKT-mTOR pathway is a novel component of oncogene induced DNA damage response in immortalized 'normal-like' breast cells and its suppression may contribute to growth arrest and arrest of the breast tumorigenesis.

  18. Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

    PubMed

    Zhao, Hong; Li, Jiangwei; Traganos, Frank; Halicka, H Dorota; Zarebski, Mirosław; Dobrucki, Jurek; Darzynkiewicz, Zbigniew

    2011-06-01

    By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

  19. Induced overexpression of CD44 associated with resistance to apoptosis on DNA damage response in human head and neck squamous cell carcinoma cells.

    PubMed

    Ohkoshi, Emika; Umemura, Naoki

    2017-02-01

    CD44 is a marker of cancer stem cells in head and neck squamous cell carcinoma, and CD44 expression is related to prognosis in cancer patients. We examined whether herbal medicine components affect CD44 expression and induce cancer cell apoptosis. Baicalin enhanced apoptosis with no effect on CD44 levels, while baicalein did not enhance apoptosis and upregulated CD44 in head and neck squamous cell carcinoma. Furthermore, baicalein induced phosphorylation of CHK1, as a marker of DNA damage response to S-to-G2/M phase arrest. Our results clearly demonstrated that baicalein enhanced expression of CD44 and accordingly enhanced the DNA damage response. These data suggest that induction of CD44 inhibited cancer cell induction of apoptosis by increasing the DNA damage response. Together, our findings suggest that CD44 expression in head and neck squamous cell carcinoma plays a role in enhancing the DNA damage response.

  20. Induced overexpression of CD44 associated with resistance to apoptosis on DNA damage response in human head and neck squamous cell carcinoma cells

    PubMed Central

    Ohkoshi, Emika; Umemura, Naoki

    2017-01-01

    CD44 is a marker of cancer stem cells in head and neck squamous cell carcinoma, and CD44 expression is related to prognosis in cancer patients. We examined whether herbal medicine components affect CD44 expression and induce cancer cell apoptosis. Baicalin enhanced apoptosis with no effect on CD44 levels, while baicalein did not enhance apoptosis and upregulated CD44 in head and neck squamous cell carcinoma. Furthermore, baicalein induced phosphorylation of CHK1, as a marker of DNA damage response to S-to-G2/M phase arrest. Our results clearly demonstrated that baicalein enhanced expression of CD44 and accordingly enhanced the DNA damage response. These data suggest that induction of CD44 inhibited cancer cell induction of apoptosis by increasing the DNA damage response. Together, our findings suggest that CD44 expression in head and neck squamous cell carcinoma plays a role in enhancing the DNA damage response. PMID:28035370

  1. Arecoline, a major alkaloid of areca nut, inhibits p53, represses DNA repair, and triggers DNA damage response in human epithelial cells.

    PubMed

    Tsai, Yi-Shan; Lee, Ka-Wo; Huang, Jau-Ling; Liu, Yu-Sen; Juo, Suh-Hang Hank; Kuo, Wen-Rei; Chang, Jan-Gowth; Lin, Chang-Shen; Jong, Yuh-Jyh

    2008-07-30

    The International Agency for Research on Cancer declared that areca nut was carcinogenic to human. Areca nut is the main component of betel quid (BQ), which is commonly consumed in Asia. Epidemiological studies have shown that BQ chewing is a predominant risk factor for oral and pharyngeal cancers. It has been known that areca nut is genotoxic to human epithelial cells. However, the molecular and cellular mechanisms underlying areca nut-associated genotoxicity are not fully understood. Here we showed that arecoline, a major alkaloid of areca nut, might contribute to oral carcinogenesis through inhibiting p53 and DNA repair. We found, on the biological aspect, that arecoline could induce gamma-H2AX phosphorylation, a sensitive DNA damage marker, in KB, HEp-2, and 293 cells, suggesting that DNA damages were elicited by arecoline. This phenomenon was supported by the observations of arecoline-induced hyperphosphorylation of ATM, Nbs1, Chk1/2, p53, and Cdc25C, as well as G2/M cell cycle arrest, indicating that a cellular DNA damage response was activated. To explore the possible mechanism accounting for arecoline-elicited DNA damages, we found that arecoline could inhibit p53 by its expression and transactivation function. As a result, the expression of p53-regulated p21(WAF1) and the p53-activated DNA repair were repressed by arecoline. Finally, we showed that p53 mRNA transcripts were frequently down-regulated in BQ-associated oral cancer, suggesting that arecoline-mediated p53 inhibition might play a role in BQ-associated tumorigenesis.

  2. NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines.

    PubMed

    Erbaykent-Tepedelen, Burcu; Ozmen, Besra; Varisli, Lokman; Gonen-Korkmaz, Ceren; Debelec-Butuner, Bilge; Muhammed Syed, Hamid; Yilmazer-Cakmak, Ozgur; Korkmaz, Kemal Sami

    2011-10-14

    NKX3.1 is an androgen-regulated homeobox gene that encodes a tissue-restricted transcription factor, which plays an important role in the differentiation of the prostate epithelium. Thus, the role of NKX3.1 as a functional topoisomerase I activity enhancer in cell cycle regulation and the DNA damage response (DDR) was explored in prostate cancer cell lines. As an early response to DNA damage following CPT-11 treatment, we found that there was an increase in the γH2AX(S139) foci number and that total phosphorylation levels were reduced in PC-3 cells following ectopic NKX3.1 expression as well as in LNCaP cells following androgen administration. Furthermore, upon drug treatment, the increase in ATM(S1981) phosphorylation was reduced in the presence of NKX3.1 expression, whereas DNA-PKcs expression was increased. Additionally, phosphorylation of CHK2(T68) and NBS1(S343) was abrogated by ectopic NKX3.1 expression, compared with the increasing levels in control PC-3 cells in a time-course experiment. Finally, NKX3.1 expression maintained a high cyclin D1 expression level regardless of drug treatment, while total γH2AX(S139) phosphorylation remained depleted in PC-3, as well as in LNCaP, cells. Thus, we suggest that androgen regulated NKX3.1 maintains an active DDR at the intra S progression and contributes to the chemotherapeutic resistance of prostate cancer cells to DNA damaging compounds.

  3. Impact of fractionation on out-of-field survival and DNA damage responses following exposure to intensity modulated radiation fields

    NASA Astrophysics Data System (ADS)

    Ghita, Mihaela; Coffey, Caroline B.; Butterworth, Karl T.; McMahon, Stephen J.; Schettino, Giuseppe; Prise, Kevin M.

    2016-01-01

    To limit toxicity to normal tissues adjacent to the target tumour volume, radiotherapy is delivered using fractionated regimes whereby the total prescribed dose is given as a series of sequential smaller doses separated by specific time intervals. The impact of fractionation on out-of-field survival and DNA damage responses was determined in AGO-1522 primary human fibroblasts and MCF-7 breast tumour cells using uniform and modulated exposures delivered using a 225 kVp x-ray source. Responses to fractionated schedules (two equal fractions delivered with time intervals from 4 h to 48 h) were compared to those following acute exposures. Cell survival and DNA damage repair measurements indicate that cellular responses to fractionated non-uniform exposures differ from those seen in uniform exposures for the investigated cell lines. Specifically, there is a consistent lack of repair observed in the out-of-field populations during intervals between fractions, confirming the importance of cell signalling to out-of-field responses in a fractionated radiation schedule, and this needs to be confirmed for a wider range of cell lines and conditions.

  4. DNA Damage, DNA Repair, Aging, and Neurodegeneration.

    PubMed

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L; Bohr, Vilhelm A

    2015-09-18

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span.

  5. p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

    PubMed

    Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

    2007-10-01

    Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

  6. Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage

    PubMed Central

    Liu, Jing; Vidi, Pierre-Alexandre; Lelièvre, Sophie A.; Irudayaraj, Joseph M. K.

    2015-01-01

    ABSTRACT Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed. PMID:25501817

  7. Regulation of mitosis in response to damaged or incompletely replicated DNA require different levels of Grapes (Drosophila Chk1)

    PubMed Central

    Purdy, Amanda; Uyetake, Lyle; Cordeiro, Melissa Garner; Su, Tin Tin

    2011-01-01

    Summary Checkpoints monitor the state of DNA and can delay or arrest the cell cycle at multiple points including G1-S transition, progress through S phase and G2-M transition. Regulation of progress through mitosis, specifically at the metaphase-anaphase transition, occurs after exposure to ionizing radiation (IR) in Drosophila and budding yeast, but has not been conclusively demonstrated in mammals. Here we report that regulation of metaphase-anaphase transition in Drosophila depends on the magnitude of radiation dose and time in the cell cycle at which radiation is applied, which may explain the apparent differences among experimental systems and offer an explanation as to why this regulation has not been seen in mammalian cells. We further document that mutants in Drosophila Chk1 (Grapes) that are capable of delaying the progress through mitosis in response to IR are incapable of delaying progress through mitosis when DNA synthesis is blocked by mutations in an essential replication factor encoded by double park (Drosophila Cdt1). We conclude that DNA damage and replication checkpoints operating in the same cell cycle at the same developmental stage in Drosophila can exhibit differential requirements for the Chk1 homolog. The converse situation exists in fission yeast where loss of Chk1 is more detrimental to the DNA damage checkpoint than to the DNA replication checkpoint. It remains to be seen which of these two different uses of Chk1 homologs are conserved in mammals. Finally, our results demonstrate that Drosophila provides a unique opportunity to study the regulation of the entry into, and progress through, mitosis by DNA structure checkpoints in metazoa. PMID:16079276

  8. DNA Damage Induced Neuronal Death

    DTIC Science & Technology

    1999-10-01

    Experiments are proposed to examine the molecular mechanism by which mustard chemical warfare agents induce neuronal cell death . DNA damage is the...proposed underlying mechanism of mustard-induced neuronal cell death . We propose a novel research strategy to test this hypothesis by using mice with...perturbed DNA repair to explore the relationship between mustard-induced DNA damage and neuronal cell death . Initial in vitro studies (Years 1, 2 & 3

  9. DNA damage response at telomeres contributes to lung aging and chronic obstructive pulmonary disease.

    PubMed

    Birch, Jodie; Anderson, Rhys K; Correia-Melo, Clara; Jurk, Diana; Hewitt, Graeme; Marques, Francisco Madeira; Green, Nicola J; Moisey, Elizabeth; Birrell, Mark A; Belvisi, Maria G; Black, Fiona; Taylor, John J; Fisher, Andrew J; De Soyza, Anthony; Passos, João F

    2015-11-15

    Cellular senescence has been associated with the structural and functional decline observed during physiological lung aging and in chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the first line of defense in the lungs and are important to COPD pathogenesis. However, the mechanisms underlying airway epithelial cell senescence, and particularly the role of telomere dysfunction in this process, are poorly understood. We aimed to investigate telomere dysfunction in airway epithelial cells from patients with COPD, in the aging murine lung and following cigarette smoke exposure. We evaluated colocalization of γ-histone protein 2A.X and telomeres and telomere length in small airway epithelial cells from patients with COPD, during murine lung aging, and following cigarette smoke exposure in vivo and in vitro. We found that telomere-associated DNA damage foci increase in small airway epithelial cells from patients with COPD, without significant telomere shortening detected. With age, telomere-associated foci increase in small airway epithelial cells of the murine lung, which is accelerated by cigarette smoke exposure. Moreover, telomere-associated foci predict age-dependent emphysema, and late-generation Terc null mice, which harbor dysfunctional telomeres, show early-onset emphysema. We found that cigarette smoke accelerates telomere dysfunction via reactive oxygen species in vitro and may be associated with ataxia telangiectasia mutated-dependent secretion of inflammatory cytokines interleukin-6 and -8. We propose that telomeres are highly sensitive to cigarette smoke-induced damage, and telomere dysfunction may underlie decline of lung function observed during aging and in COPD.

  10. TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons.

    PubMed

    Zhang, Peisu; Furukawa, Katsutoshi; Opresko, Patricia L; Xu, Xiangru; Bohr, Vilhelm A; Mattson, Mark P

    2006-04-01

    Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.

  11. DNA damage response in renal ischemia-reperfusion and ATP-depletion injury of renal tubular cells.

    PubMed

    Ma, Zhengwei; Wei, Qingqing; Dong, Guie; Huo, Yuqing; Dong, Zheng

    2014-07-01

    Renal ischemia-reperfusion leads to acute kidney injury (AKI) that is characterized pathologically by tubular damage and cell death, followed by tubular repair, atrophy and interstitial fibrosis. Recent work suggested the possible presence of DNA damage response (DDR) in AKI. However, the evidence is sketchy and the role and regulation of DDR in ischemic AKI remain elusive. In this study, we demonstrated the induction of phosphorylation of ATM, H2AX, Chk2 and p53 during renal ischemia-reperfusion in mice, suggesting DDR in kidney tissues. DDR was also induced in vitro during the recovery or "reperfusion" of renal proximal tubular cells (RPTCs) after ATP depletion. DDR in RPTCs was abrogated by supplying glucose to maintain ATP via glycolysis, indicating that the DDR depends on ATP depletion. The DDR was also suppressed by the general caspase inhibitor z-VAD and the overexpression of Bcl-2, supporting a role of apoptosis-associated DNA damage in the DDR. N-acetylcysteine (NAC), an antioxidant, suppressed the phosphorylation of ATM and p53 and, to a less extent, Chk2, but NAC increased the phosphorylation and nuclear foci formation of H2AX. Interestingly, NAC increased apoptosis, which may account for the observed H2AX activation. Ku55933, an ATM inhibitor, blocked ATM phosphorylation and ameliorated the phosphorylation of Chk2 and p53, but it increased H2AX phosphorylation and nuclear foci formation. Ku55933 also increased apoptosis in RPTCs following ATP depletion. The results suggest that DDR occurs during renal ischemia-reperfusion in vivo and ATP-depletion injury in vitro. The DDR is partially induced by apoptosis and oxidative stress-related DNA damage. ATM, as a sensor in the DDR, may play a cytoprotective role against tubular cell injury and death.

  12. CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA-binding proteins which trigger host DNA damage response.

    PubMed

    Ramirez-Garcés, Diana; Camborde, Laurent; Pel, Michiel J C; Jauneau, Alain; Martinez, Yves; Néant, Isabelle; Leclerc, Catherine; Moreau, Marc; Dumas, Bernard; Gaulin, Elodie

    2016-04-01

    To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR.

  13. APC/C(Cdh1) controls CtIP stability during the cell cycle and in response to DNA damage.

    PubMed

    Lafranchi, Lorenzo; de Boer, Harmen R; de Vries, Elisabeth G E; Ong, Shao-En; Sartori, Alessandro A; van Vugt, Marcel A T M

    2014-12-01

    Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C(C) (dh1) ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is currently unknown whether APC/C(C) (dh1) also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA-damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA-end resection factor, as a novel APC/C(C) (dh1) target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G1 and after DNA damage in G2. Finally, we find that abrogating the CtIP-Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA-end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/C(C) (dh1) on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner.

  14. Knockdown of Minichromosome Maintenance Proteins Inhibits Foci Forming of Mediator of DNA-Damage Checkpoint 1 in Response to DNA Damage in Human Esophageal Squamous Cell Carcinoma TE-1 Cells.

    PubMed

    Yu, Jinzhong; Wang, Ruijie; Wu, Jinfeng; Dang, Zhongqin; Zhang, Qinsheng; Li, Bo

    2016-10-01

    Esophageal squamous cell carcinoma (ESCC) has a high morbidity in China and its treatment depends greatly on adjuvant chemotherapy. However, DNA damage repair in cancer cells severely affects the outcome of treatment. This study investigated the potential mechanism regarding mediator of DNA-damage checkpoint 1 (MDC1) and minichromosome maintenance proteins (MCMs) during DNA damage in ESCC. Recombinant vectors of MDC1 and MCMs with tags were constructed and transfected into human ESCC cell line TE-1. Immunoprecipitation and mass spectrometry were performed to screen the MCMs interacting with MDC1, and direct interaction was confirmed by glutathione S-transferase (GST) pull-down assay in vitro. MCM2 and MCM6 were knocked down by shRNAs, after which chromatin fraction and foci forming of MDC1 upon bleomycin-induced DNA damage were examined. The results showed that MCM2/3/5/6 were immunoprecipitated by antibodies against the tag of MDC1 in TE-1 nuclei, and the GST pull-down assay indicated the direct interaction. Knockdown of MCM2 or MCM6 reduced the chromatin fraction of MDC1 according to Western blot results. Moreover, knockdown of MCM2 or MCM6 could significantly inhibit foci forming of MDC1 in TE-1 nuclei in response to bleomycin-induced DNA damage (p < 0.001). This study indicates the direct interaction between MDC1 and MCMs in TE-1 nuclei. Downregulation of MCMs can inhibit chromatin fraction and foci forming of MDC1 in TE-1 cells upon DNA damage, which suggests MCMs and MDC1 as potential targets to improve the outcome of chemotherapy in ESCC.

  15. Surviving the breakup: the DNA damage checkpoint.

    PubMed

    Harrison, Jacob C; Haber, James E

    2006-01-01

    In response to even a single chromosomal double-strand DNA break, cells enact the DNA damage checkpoint. This checkpoint triggers cell cycle arrest, providing time for the cell to repair damaged chromosomes before entering mitosis. This mechanism helps prevent the segregation of damaged or mutated chromosomes and thus promotes genomic stability. Recent work has elucidated the molecular mechanisms underlying several critical steps in checkpoint activation, notably the recruitment of the upstream checkpoint kinases of the ATM and ATR families to different damaged DNA structures and the molecular events through which these kinases activate their effectors. Chromatin modification has emerged as one important component of checkpoint activation and maintenance. Following DNA repair, the checkpoint pathway is inactivated in a process termed recovery. A related but genetically distinct process, adaptation, controls cell cycle re-entry in the face of unrepairable damage.

  16. Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway.

    PubMed

    Faucher, David; Wellinger, Raymund J

    2010-08-26

    Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3), both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB) repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells.

  17. Genetic Control or Repair and Adaptive Response to Low-Level DNA Damage

    SciTech Connect

    J. E. Haber

    2009-10-05

    Research was focused on how a single double-strand break - a model of low-dose ionizing radiation-induced DNA damage - could be studied in a simple model system, budding yeast. Breaks were induced in several different ways. We used the site-specific HO endonuclease to create a single DSB in all cells of the population so that its fate could be extensively analyzed genetically and molecularly. We also used two heterologous systems, the plant DS element and the Rag1/Rag2 proteins, to generate different types of DSBs, these containing hairpin ends that needed to be cleaved open before end-joining could take place. All three approaches yielded important new findings. We also extended our analysis of the Mre11 protein that plays key roles in both NHEJ and in homologous recombination. Finally we analyzed the poorly understood recombination events that were independent of the key recombination protein, Rad52. This line of inquiry was strongly motivated by the fact that vertebrate cells do not rely strongly on Rad52 for homologous recombination, so that some clues about alternative mechanisms could be gained by understanding how Rad52-independent recombination occurred. We found that the Mre11 complex was the most important element in Rad52-independent recombination.

  18. DNA damage response (DDR) induced by topoisomerase II poisons requires nuclear function of the small GTPase Rac.

    PubMed

    Wartlick, Friedrich; Bopp, Anita; Henninger, Christian; Fritz, Gerhard

    2013-12-01

    Here, we investigated the influence of Rac family small GTPases on mechanisms of the DNA damage response (DDR) stimulated by topoisomerase II poisons. To this end, we examined the influence of the Rac-specific small molecule inhibitor EHT1864 on Ser139 phosphorylation of histone H2AX, a widely used marker of the DDR triggered by DNA double-strand breaks. EHT1864 attenuated the doxorubicin-stimulated DDR in a subset of cell lines tested, including HepG2 hepatoma cells. EHT1864 reduced the level of DNA strand breaks and increased viability following treatment of HepG2 cells with topo II poisons. Protection by EHT1864 was observed in both p53 wildtype (HepG2) and p53 deficient (Hep3B) human hepatoma cells and, furthermore, remained unaffected upon pharmacological inhibition of p53 in HepG2. Apparently, the impact of Rac on the DDR is independent of p53. Protection from doxorubicin-induced DNA damage by EHT1864 comprises both S and G2 phase cells. The inhibitory effect of EHT1864 on doxorubicin-stimulated DDR was mimicked by pharmacological inhibition of various protein kinases, including JNK, ERK, PI3K, PAK and CK1. EHT1864 and protein kinase inhibitors also attenuated the formation of the topo II-DNA cleavable complex. Moreover, EHT1864 mitigated the constitutive phosphorylation of topoisomerase IIα at positions S1106, S1213 and S1247. Doxorubicin transport, nuclear import/export of topoisomerase II and Hsp90-related mechanisms are likely not of relevance for doxorubicin-stimulated DDR impaired by EHT1864. We suggest that multiple kinase-dependent but p53- and heat shock protein-independent Rac-regulated nuclear mechanisms are required for activation of the DDR following treatment with topo II poisons.

  19. Topoisomerase II Inhibitors Induce DNA Damage-Dependent Interferon Responses Circumventing Ebola Virus Immune Evasion

    PubMed Central

    Luthra, Priya; Aguirre, Sebastian; Yen, Benjamin C.; Pietzsch, Colette A.; Sanchez-Aparicio, Maria T.; Tigabu, Bersabeh; Morlock, Lorraine K.; García-Sastre, Adolfo; Leung, Daisy W.; Williams, Noelle S.; Fernandez-Sesma, Ana; Bukreyev, Alexander

    2017-01-01

    ABSTRACT Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication. PMID:28377530

  20. Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response

    PubMed Central

    Yang, Seungchan; Quaresma, Alexandre J. C.; Nickerson, Jeffrey A.; Green, Karin M.; Shaffer, Scott A.; Imbalzano, Anthony N.; Martin-Buley, Lori A.; Lian, Jane B.; Stein, Janet L.; van Wijnen, Andre J.; Stein, Gary S.

    2015-01-01

    ABSTRACT Cancer cells exhibit modifications in nuclear architecture and transcriptional control. Tumor growth and metastasis are supported by RUNX family transcriptional scaffolding proteins, which mediate the assembly of nuclear-matrix-associated gene-regulatory hubs. We used proteomic analysis to identify RUNX2-dependent protein–protein interactions associated with the nuclear matrix in bone, breast and prostate tumor cell types and found that RUNX2 interacts with three distinct proteins that respond to DNA damage – RUVBL2, INTS3 and BAZ1B. Subnuclear foci containing these proteins change in intensity or number following UV irradiation. Furthermore, RUNX2, INTS3 and BAZ1B form UV-responsive complexes with the serine-139-phosphorylated isoform of H2AX (γH2AX). UV irradiation increases the interaction of BAZ1B with γH2AX and decreases histone H3 lysine 9 acetylation levels, which mark accessible chromatin. RUNX2 depletion prevents the BAZ1B–γH2AX interaction and attenuates loss of H3K9 and H3K56 acetylation. Our data are consistent with a model in which RUNX2 forms functional complexes with BAZ1B, RUVBL2 and INTS3 to mount an integrated response to DNA damage. This proposed cytoprotective function for RUNX2 in cancer cells might clarify its expression in chemotherapy-resistant and/or metastatic tumors. PMID:25609707

  1. Cleavage Factor I Links Transcription Termination to DNA Damage Response and Genome Integrity Maintenance in Saccharomyces cerevisiae

    PubMed Central

    Gaillard, Hélène; Aguilera, Andrés

    2014-01-01

    During transcription, the nascent pre-mRNA undergoes a series of processing steps before being exported to the cytoplasm. The 3′-end processing machinery involves different proteins, this function being crucial to cell growth and viability in eukaryotes. Here, we found that the rna14-1, rna15-1, and hrp1-5 alleles of the cleavage factor I (CFI) cause sensitivity to UV-light in the absence of global genome repair in Saccharomyces cerevisiae. Unexpectedly, CFI mutants were proficient in UV-lesion repair in a transcribed gene. DNA damage checkpoint activation and RNA polymerase II (RNAPII) degradation in response to UV were delayed in CFI-deficient cells, indicating that CFI participates in the DNA damage response (DDR). This is further sustained by the synthetic growth defects observed between rna14-1 and mutants of different repair pathways. Additionally, we found that rna14-1 suffers severe replication progression defects and that a functional G1/S checkpoint becomes essential in avoiding genetic instability in those cells. Thus, CFI function is required to maintain genome integrity and to prevent replication hindrance. These findings reveal a new function for CFI in the DDR and underscore the importance of coordinating transcription termination with replication in the maintenance of genomic stability. PMID:24603480

  2. Intense THz pulses cause H2AX phosphorylation and activate DNA damage response in human skin tissue

    PubMed Central

    Titova, Lyubov V.; Ayesheshim, Ayesheshim K.; Golubov, Andrey; Fogen, Dawson; Rodriguez-Juarez, Rocio; Hegmann, Frank A.; Kovalchuk, Olga

    2013-01-01

    Recent emergence and growing use of terahertz (THz) radiation for medical imaging and public security screening raise questions on reasonable levels of exposure and health consequences of this form of electromagnetic radiation. In particular, picosecond-duration THz pulses have shown promise for novel diagnostic imaging techniques. However, the effects of THz pulses on human cells and tissues thus far remain largely unknown. We report on the investigation of the biological effects of pulsed THz radiation on artificial human skin tissues. We observe that exposure to intense THz pulses for ten minutes leads to a significant induction of H2AX phosphorylation, indicating that THz pulse irradiation may cause DNA damage in exposed skin tissue. At the same time, we find a THz-pulse-induced increase in the levels of several proteins responsible for cell-cycle regulation and tumor suppression, suggesting that DNA damage repair mechanisms are quickly activated. Furthermore, we find that the cellular response to pulsed THz radiation is significantly different from that induced by exposure to UVA (400 nm). PMID:23577291

  3. Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells.

    PubMed

    Morelli, Maria Beatrice; Amantini, Consuelo; Santoni, Matteo; Soriani, Alessandra; Nabissi, Massimo; Cardinali, Claudio; Santoni, Angela; Santoni, Giorgio

    2015-11-03

    Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC.Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy.On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner.Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity.

  4. Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells

    PubMed Central

    Morelli, Maria Beatrice; Amantini, Consuelo; Santoni, Matteo; Soriani, Alessandra; Nabissi, Massimo; Cardinali, Claudio; Santoni, Angela; Santoni, Giorgio

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC. Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy. On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner. Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity. PMID:26474283

  5. gammaH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products.

    PubMed

    Albino, Anthony P; Jorgensen, Ellen D; Rainey, Patrick; Gillman, Gene; Clark, T Jeffrey; Gietl, Diana; Zhao, Hong; Traganos, Frank; Darzynkiewicz, Zbigniew

    2009-08-01

    Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, it would be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenic DNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previously we showed that phosphorylation of H2AX (defined as gammaH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that gammaH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring gammaH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that gammaH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that gammaH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.

  6. Adeno-associated virus type 2 modulates the host DNA damage response induced by herpes simplex virus 1 during coinfection.

    PubMed

    Vogel, Rebecca; Seyffert, Michael; Strasser, Regina; de Oliveira, Anna P; Dresch, Christiane; Glauser, Daniel L; Jolinon, Nelly; Salvetti, Anna; Weitzman, Matthew D; Ackermann, Mathias; Fraefel, Cornel

    2012-01-01

    Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.

  7. Optical detection of DNA damage

    NASA Astrophysics Data System (ADS)

    Rogers, Kim R.; Apostol, A.; Cembrano, J.

    1999-02-01

    A rapid and sensitive fluorescence assay for oxidative damage to calf thymus DNA is reported. A decrease in the transition temperature for strand separation resulted from exposure of the DNA to the reactive decomposition products of 3- morpholinosydnonimine (SIN-1) (i.e., nitric oxide, superoxide, peroxynitrite, hydrogen peroxide, and hydroxyl radicals). A decrease in melting temperature of 12 degrees Celsius was indicative of oxidative damage including single strand chain breaks. Double stranded (ds) and single stranded (ss) forms of DNA were determined using the indicator dyes ethidium bromide and PicoGreen. The change in DNA 'melting' curves was dependant on the concentration of SIN-1 and was most pronounced at 75 degrees Celsius. This chemically induced damage was significantly inhibited by sodium citrate, tris(hydroxymethyl)aminomethane (Tris), and diethylenetriaminepentaacetic acid (DTPA), but was unaffected by superoxide dismutase (SOD), catalase, ethylenediamine tetraacietic acid (EDTA), or deferoxamine. Lowest observable effect level for SIN-1-induced damage was 200 (mu) M.

  8. The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

    PubMed

    Du, W; Amarachintha, S; Wilson, A; Pang, Q

    2017-02-01

    Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca(-/-) hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca(-/-) pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca(-/-) pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca(-/-) pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

  9. Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

    PubMed

    Akaike, Y; Kuwano, Y; Nishida, K; Kurokawa, K; Kajita, K; Kano, S; Masuda, K; Rokutan, K

    2015-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent

  10. A Transposable Element within the Non-canonical Telomerase RNA of Arabidopsis thaliana Modulates Telomerase in Response to DNA Damage

    PubMed Central

    Xu, Hengyi; Nelson, Andrew D. L.; Shippen, Dorothy E.

    2015-01-01

    Long noncoding RNAs (lncRNAs) have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs). TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3’ terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE). DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2) and without DRE (TER2Δ) we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault. PMID:26075395

  11. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

    PubMed

    Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

    2015-03-01

    Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

  12. The Aspergillus nidulans uvsB gene encodes an ATM-related kinase required for multiple facets of the DNA damage response.

    PubMed Central

    Hofmann, A F; Harris, S D

    2000-01-01

    In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response. PMID:10747054

  13. Assessment of DNA damage and molecular responses in Labeo rohita (Hamilton, 1822) following short-term exposure to silver nanoparticles.

    PubMed

    Sharma, Niti; Rather, Mohd Ashraf; Ajima, Malachy N O; Gireesh-Babu, P; Kumar, Kundan; Sharma, Rupam

    2016-10-01

    The increasing application of silver nanoparticles (Ag-NPs) both in industries and in agricultural fields has led to its accumulation in the aquatic ecosystem through water run-off. In the present study, the effects of Ag-NPs in the liver of Labeo rohita, were investigated at genomic and cellular level for seven days at the concentrations of 100, 200, 400 and 800 μg l(-1) by using 18 and 29 nm sizes of Ag-NPs. The Ag-NPs sizes of 18 and 29 nm were synthesized by a chemical method using atomic force microscopy with the zeta potential of -55 mV and-31.4 mV respectively. They were found to be spherical with smooth surfaces. Assessment of genotoxic effects of the particles in the fish using single-cell gel electrophoresis showed DNA damage on exposure to concentrations of 400 and 800 μg l(-1). Histopathological examination of the liver revealed vacuolar degeneration, hepatocytes have undergone total degeneration and high accumulation of Ag-NPs that depicted both time and dose-dependent relationships. Furthermore, the expression study of stress-related genes showed down-regulation, due to the production of free radicals and reactive oxygen species. Ag-NPs can cause both DNA damage and affect the cellular responses of L. rohita.

  14. RAD51B Activity and Cell Cycle Regulation in Response to DNA Damage in Breast Cancer Cell Lines

    PubMed Central

    Lee, Phoebe S; Fang, Jun; Jessop, Lea; Myers, Timothy; Raj, Preethi; Hu, Nan; Wang, Chaoyu; Taylor, Philip R; Wang, Jianjun; Khan, Javed; Jasin, Maria; Chanock, Stephen J

    2014-01-01

    Common genetic variants mapping to two distinct regions of RAD51B, a paralog of RAD51, have been associated with breast cancer risk in genome-wide association studies (GWAS). RAD51B is a plausible candidate gene because of its established role in the homologous recombination (HR) process. How germline genetic variation in RAD51B confers susceptibility to breast cancer is not well understood. Here, we investigate the molecular function of RAD51B in breast cancer cell lines by knocking down RAD51B expression by small interfering RNA and treating cells with DNA-damaging agents, namely cisplatin, hydroxyurea, or methyl-methanesulfonate. Our results show that RAD51B-depleted breast cancer cells have increased sensitivity to DNA damage, reduced efficiency of HR, and altered cell cycle checkpoint responses. The influence of RAD51B on the cell cycle checkpoint is independent of its role in HR and further studies are required to determine whether these functions can explain the RAD51B breast cancer susceptibility alleles. PMID:25368520

  15. DNA damage response (DDR) and senescence: shuttled inflamma-miRNAs on the stage of inflamm-aging.

    PubMed

    Olivieri, Fabiola; Albertini, Maria Cristina; Orciani, Monia; Ceka, Artan; Cricca, Monica; Procopio, Antonio Domenico; Bonafè, Massimiliano

    2015-11-03

    A major issue in aging research is how cellular phenomena affect aging at the systemic level. Emerging evidence suggests that DNA damage response (DDR) signaling is a key mechanism linking DNA damage accumulation, cell senescence, and organism aging. DDR activation in senescent cells promotes acquisition of a proinflammatory secretory phenotype (SASP), which in turn elicits DDR and SASP activation in neighboring cells, thereby creating a proinflammatory environment extending at the local and eventually the systemic level. DDR activation is triggered by genomic lesions as well as emerging bacterial and viral metagenomes. Therefore, the buildup of cells with an activated DDR probably fuels inflamm-aging and predisposes to the development of the major age-related diseases (ARDs). Micro (mi)-RNAs - non-coding RNAs involved in gene expression modulation - are released locally and systemically by a variety of shuttles (exosomes, lipoproteins, proteins) that likely affect the efficiency of their biological effects. Here we suggest that some miRNAs, previously found to be associated with inflammation and senescence - miR-146, miR-155, and miR-21 - play a central role in the interplay among DDR, cell senescence and inflamm-aging. The identification of the functions of shuttled senescence-associated miRNAs is expected to shed light on the aging process and on how to delay ARD development.

  16. Molecular responses to photogenotoxic stress induced by the antibiotic lomefloxacin in human skin cells: from DNA damage to apoptosis.

    PubMed

    Marrot, Laurent; Belaïdi, Jean Phillipe; Jones, Christophe; Perez, Phillipe; Riou, Lydia; Sarasin, Alain; Meunier, Jean Roch

    2003-09-01

    Photo-unstable chemicals sometimes behave as phototoxins in skin, inducing untoward clinical side-effects when exposed to sunlight. Some drugs, such as psoralens or fluoroquinolones, can damage genomic DNA, thus increasing the risk of photocarcinogenesis. Here, lomefloxacin, an antibiotic from the fluoroquinolone family known to be involved in skin tumor development in photoexposed mice, was studied using normal human skin cells in culture: fibroblasts, keratinocytes, and Caucasian melanocytes. When treated cells were exposed to simulated solar ultraviolet A (320-400 nm), lomefloxacin induced damage such as strand breaks and pyrimidine dimers in genomic DNA. Lomefloxacin also triggered various stress responses: heme-oxygenase-1 expression in fibroblasts, changes in p53 status as shown by the accumulation of p53 and p21 proteins or the induction of MDM2 and GADD45 genes, and stimulation of melanogenesis by increasing the tyrosinase activity in melanocytes. Lomefloxacin could also lead to apoptosis in keratinocytes exposed to ultraviolet A: caspase-3 was activated and FAS-L gene was induced. Moreover, keratinocytes were shown to be the most sensitive cell type to lomefloxacin phototoxic effects, in spite of the well-established effectiveness of their antioxidant equipment. These data show that the phototoxicity of a given drug can be driven by different mechanisms and that its biologic impact varies according to cell type.

  17. Comparative effects of Cd and Pb on biochemical response and DNA damage in the earthworm Eisenia fetida (Annelida, Oligochaeta).

    PubMed

    Li, Mei; Liu, Zhengtao; Xu, Yun; Cui, Yibin; Li, Dingsheng; Kong, Zhiming

    2009-02-01

    There are rising concerns about the hazardous effects of cadmium (Cd) and lead (Pb) on the environment in China. Biochemical and comet assays were conducted on the earthworm Eisenia fetida, a suitable bio-indicator organism for evaluating soil pollution after exposure to two heavy metals, Cd and Pb. Protein content increased at low Cd concentrations (p<0.05) and decreased at the highest concentration of 10 mg kg(-1), compared to control (p<0.05). Pb showed an inhibitory effect on protein content at low concentrations but demonstrated no significant effect at higher concentrations. There were no significant differences between control and treated groups at the doses of 1 and 10 mg kg(-1) Cd while at a dose of 0.1 mg kg(-1) Cd the cellulase activity was significantly increased compared to control. Cellulase activities of Pb-treated E. fetida increased in a dose dependent fashion. Results of the comet assay indicated toxicant induced DNA damage. Cd exposure caused significant differences between control and treatment groups (ANOVA, p< 0.05, p< 0.01) and a positive dose-response profile. As for Pb treatment, there were no significant differences between the groups treated with 50 and 500 mg kg(-1) of Pb and the control. Results showed that DNA damage from Cd was more serious than that from Pb. And this indicated that the earthworm was more sensitive to the effects of Cd.

  18. DNA Repair by Reversal of DNA Damage

    PubMed Central

    Yi, Chengqi; He, Chuan

    2013-01-01

    Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology. PMID:23284047

  19. 'Fukusensor:' a genetically engineered plant for reporting DNA damage in response to gamma radiation.

    PubMed

    Peng, Yanhui; Allen, Sara; Millwood, Reginald J; Stewart, C Neal

    2014-12-01

    Transgenic plants can be designed to be 'phytosensors' for detection of environmental contaminants and pathogens. In this study, we describe the design and testing of a radiation phytosensor in the form of green fluorescence protein (GFP)-transgenic Arabidopsis plant utilizing a DNA repair deficiency mutant background as a host. Mutant lines of Arabidopsis AtATM (At3g48190), which are hypersensitive to gamma irradiation, were used to generate stable GFP transgenic plants in which a gfp gene was under the control of a strong constitutive CaMV 35S promoter. Mutant and nonmutant genetic background transgenic plants were treated with 0, 1, 5, 10 and 100 Gy radiation doses, respectively, using a Co-60 source. After 1 week, the GFP expression levels were drastically reduced in young leaves of mutant background plants (treated by 10 and 100 Gy), whereas there were scant visible differences in the fluorescence of the nonmutant background plants. These early results indicate that transgenic plants could serve in a relevant sensor system to report radiation dose and the biological effects to organisms in response to radionuclide contamination.

  20. Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment

    PubMed Central

    2016-01-01

    ABSTRACT There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized. PMID:27624129

  1. ErbB4 localization to cardiac myocyte nuclei, and its role in myocyte DNA damage response

    SciTech Connect

    Icli, Basak; Bharti, Ajit; Pentassuglia, Laura; Peng, Xuyang; Sawyer, Douglas B.

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer ErbB4 localizes to cardiac myocyte nuclei as a full-length receptor. Black-Right-Pointing-Pointer Cardiac myocytes express predominantly JM-a/CYT-1 ErbB4. Black-Right-Pointing-Pointer Myocyte p53 activation in response to doxorubicin requires ErbB4 activity. -- Abstract: The intracellular domain of ErbB4 receptor tyrosine kinase is known to translocate to the nucleus of cells where it can regulate p53 transcriptional activity. The purpose of this study was to examine whether ErbB4 can localize to the nucleus of adult rat ventricular myocytes (ARVM), and regulate p53 in these cells. We demonstrate that ErbB4 does locate to the nucleus of cardiac myocytes as a full-length protein, although nuclear location occurs as a full-length protein that does not require Protein Kinase C or {gamma}-secretase activity. Consistent with this we found that only the non-cleavable JM-b isoform of ErbB4 is expressed in ARVM. Doxorubicin was used to examine ErbB4 role in regulation of a DNA damage response in ARVM. Doxorubicin induced p53 and p21 was suppressed by treatment with AG1478, an EGFR and ErbB4 kinase inhibitor, or suppression of ErbB4 expression with small interfering RNA. Thus ErbB4 localizes to the nucleus as a full-length protein, and plays a role in the DNA damage response induced by doxorubicin in cardiac myocytes.

  2. Molecular responses as indicators of marine pollution: DNA damage and enzyme induction in Limanda limanda and Asterias rubens

    SciTech Connect

    Everaarts, J.M.; Sleiderink, H.M.; Besten, P.J. den

    1994-12-01

    During a survey from 26 August through 13 September 1991, specimens of the flatfish, Limanda limanda (dab), and the asteroid echinoderm, Asterias rubens (seastar), were collected at sampling locations along transacts radiating into the North Sea from the coastal zone of The Netherlands. In homogenates of liver tissue from male dab and the digestive gland (pyloric caeca) of female seastar, DNA damage (strand breaks) and induction of the cytochrome P450-dependent monooxygenase system (MO) were determined. Areas could be described with significantly increased percentages of strand breaks (lower integrity) both in dab and seastar. However, enhanced DNA strand breaks did not correspond with contamination gradients, expressed as concentrations of polychlorinated biphenyls (PCBs) or polyaromatic hydrocarbons. MO enzyme induction in the hepatic 13,000g fraction of male dab, measured as 7-ethoxyresorufin-O-deethylase activity, was significantly enhanced in response to low ambient temperatures. Some evidence was found for the facilitation of benzo[a]pyrene hydroxylase activity expressing the enzyme induction in the microsomal fraction of pyloric caeca of seastars, at increasing PCB concentrations. DNA integrity and enzyme induction elucidate the physiologic status and might be indicative for ambient impairment within restricted areas, and not necessarily related to the presence of anthropogenic or xeno-biotic substances. 27 refs., 5 figs., 1 tab.

  3. Sperm DNA oxidative damage and DNA adducts

    PubMed Central

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  4. Human parvovirus B19 DNA replication induces a DNA damage response that is dispensable for cell cycle arrest at phase G2/M.

    PubMed

    Lou, Sai; Luo, Yong; Cheng, Fang; Huang, Qinfeng; Shen, Weiran; Kleiboeker, Steve; Tisdale, John F; Liu, Zhengwen; Qiu, Jianming

    2012-10-01

    Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.

  5. Comprehensive SNP scan of DNA repair and DNA damage response genes reveal multiple susceptibility loci conferring risk to tobacco associated leukoplakia and oral cancer.

    PubMed

    Mondal, Pinaki; Datta, Sayantan; Maiti, Guru Prasad; Baral, Aradhita; Jha, Ganga Nath; Panda, Chinmay Kumar; Chowdhury, Shantanu; Ghosh, Saurabh; Roy, Bidyut; Roychoudhury, Susanta

    2013-01-01

    Polymorphic variants of DNA repair and damage response genes play major role in carcinogenesis. These variants are suspected as predisposition factors to Oral Squamous Cell Carcinoma (OSCC). For identification of susceptible variants affecting OSCC development in Indian population, the "maximally informative" method of SNP selection from HapMap data to non-HapMap populations was applied. Three hundred twenty-five SNPs from 11 key genes involved in double strand break repair, mismatch repair and DNA damage response pathways were genotyped on a total of 373 OSCC, 253 leukoplakia and 535 unrelated control individuals. The significantly associated SNPs were validated in an additional cohort of 144 OSCC patients and 160 controls. The rs12515548 of MSH3 showed significant association with OSCC both in the discovery and validation phases (discovery P-value: 1.43E-05, replication P-value: 4.84E-03). Two SNPs (rs12360870 of MRE11A, P-value: 2.37E-07 and rs7003908 of PRKDC, P-value: 7.99E-05) were found to be significantly associated only with leukoplakia. Stratification of subjects based on amount of tobacco consumption identified SNPs that were associated with either high or low tobacco exposed group. The study reveals a synergism between associated SNPs and lifestyle factors in predisposition to OSCC and leukoplakia.

  6. Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response

    PubMed Central

    Shanle, Erin K.; Andrews, Forest H.; Meriesh, Hashem; McDaniel, Stephen L.; Dronamraju, Raghuvar; DiFiore, Julia V.; Jha, Deepak; Wozniak, Glenn G.; Bridgers, Joseph B.; Kerschner, Jenny L.; Krajewski, Krzysztof; Martín, Glòria Mas; Morrison, Ashby J.; Kutateladze, Tatiana G.; Strahl, Brian D.

    2015-01-01

    The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14. PMID:26341557

  7. A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response.

    PubMed

    Alpatov, Roman; Lesch, Bluma J; Nakamoto-Kinoshita, Mika; Blanco, Andres; Chen, Shuzhen; Stützer, Alexandra; Armache, Karim J; Simon, Matthew D; Xu, Chao; Ali, Muzaffar; Murn, Jernej; Prisic, Sladjana; Kutateladze, Tatiana G; Vakoc, Christopher R; Min, Jinrong; Kingston, Robert E; Fischle, Wolfgang; Warren, Stephen T; Page, David C; Shi, Yang

    2014-05-08

    Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.

  8. The DNA damage response and immune signaling alliance: Is it good or bad? Nature decides when and where.

    PubMed

    Pateras, Ioannis S; Havaki, Sophia; Nikitopoulou, Xenia; Vougas, Konstantinos; Townsend, Paul A; Panayiotidis, Michalis I; Georgakilas, Alexandros G; Gorgoulis, Vassilis G

    2015-10-01

    The characteristic feature of healthy living organisms is the preservation of homeostasis. Compelling evidence highlight that the DNA damage response and repair (DDR/R) and immune response (ImmR) signaling networks work together favoring the harmonized function of (multi)cellular organisms. DNA and RNA viruses activate the DDR/R machinery in the host cells both directly and indirectly. Activation of DDR/R in turn favors the immunogenicity of the incipient cell. Hence, stimulation of DDR/R by exogenous or endogenous insults triggers innate and adaptive ImmR. The immunogenic properties of ionizing radiation, a prototypic DDR/R inducer, serve as suitable examples of how DDR/R stimulation alerts host immunity. Thus, critical cellular danger signals stimulate defense at the systemic level and vice versa. Disruption of DDR/R-ImmR cross talk compromises (multi)cellular integrity, leading to cell-cycle-related and immune defects. The emerging DDR/R-ImmR concept opens up a new avenue of therapeutic options, recalling the Hippocrates quote "everything in excess is opposed by nature."

  9. Types and Consequences of DNA Damage

    EPA Science Inventory

    This review provides a concise overview of the types of DNA damage and the molecular mechanisms by which a cell senses DNA damage, repairs the damage, converts the damage into a mutation, or dies as a consequence of unrepaired DNA damage. Such information is important in consid...

  10. DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

    NASA Astrophysics Data System (ADS)

    Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

    2013-11-01

    Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

  11. Inhibition of Polo-like kinase 1 during the DNA damage response is mediated through loss of Aurora A recruitment by Bora.

    PubMed

    Bruinsma, W; Aprelia, M; García-Santisteban, I; Kool, J; Xu, Y J; Medema, R H

    2016-10-10

    When cells in G2 phase are challenged with DNA damage, several key mitotic regulators such as Cdk1/Cyclin B, Aurora A and Plk1 are inhibited to prevent entry into mitosis. Here we have studied how inhibition of Plk1 is established after DNA damage. Using a Förster resonance energy transfer (FRET)-based biosensor for Plk1 activity, we show that inhibition of Plk1 after DNA damage occurs with relatively slow kinetics and is entirely dependent on loss of Plk1-T210 phosphorylation. As T210 is phosphorylated by the kinase Aurora A in conjunction with its co-factor Bora, we investigated how they are affected by DNA damage. Interestingly, we find that the interaction between Bora and Plk1 remains intact during the early phases of the DNA damage response (DDR), whereas Plk1 activity is already inhibited at this stage. Expression of an Aurora A mutant that is refractory to inhibition by the DDR failed to prevent inhibition of Plk1 and loss of T210 phosphorylation, suggesting that inhibition of Plk1 may be established by perturbing recruitment of Aurora A by Bora. Indeed, expression of a fusion in which Aurora A was directly coupled to Bora prevented DNA damage-induced inhibition of Plk1 activity, as well as inhibition of T210 phosphorylation. Taken together, these data demonstrate that DNA damage affects the function of Aurora A at multiple levels: both by direct inhibition of Aurora A activity, as well as by perturbing the interaction with its co-activator Bora. We propose that the DDR targets recruitment of Aurora A to the Plk1/Bora complex to prevent activation of Plk1 during DNA damage in G2.Oncogene advance online publication, 10 October 2016; doi:10.1038/onc.2016.347.

  12. Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups

    PubMed Central

    Licursi, Valerio; Brito, Catarina; La Torre, Mattia; Alves, Paula M.; Simao, Daniel; Mottini, Carla; Salinas, Sara; Negri, Rodolfo; Tagliafico, Enrico; Kremer, Eric J.; Saggio, Isabella

    2015-01-01

    Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR. PMID:26207738

  13. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

    SciTech Connect

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-07-01

    Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.

  14. DNA Damage and Repair in Vascular Disease.

    PubMed

    Uryga, Anna; Gray, Kelly; Bennett, Martin

    2016-01-01

    DNA damage affecting both genomic and mitochondrial DNA is present in a variety of both inherited and acquired vascular diseases. Multiple cell types show persistent DNA damage and a range of lesions. In turn, DNA damage activates a variety of DNA repair mechanisms, many of which are activated in vascular disease. Such DNA repair mechanisms either stall the cell cycle to allow repair to occur or trigger apoptosis or cell senescence to prevent propagation of damaged DNA. Recent evidence has indicated that DNA damage occurs early, is progressive, and is sufficient to impair function of cells composing the vascular wall. The consequences of persistent genomic and mitochondrial DNA damage, including inflammation, cell senescence, and apoptosis, are present in vascular disease. DNA damage can thus directly cause vascular disease, opening up new possibilities for both prevention and treatment. We review the evidence for and the causes, types, and consequences of DNA damage in vascular disease.

  15. Regulation of ATM-Dependent DNA Damage Responses in Breast Cancer by the RhoGEF Net1

    DTIC Science & Technology

    2014-04-01

    later the cells were tested for DNA damage by comet assay. Shown is a representative experiment from three independent experiments. (B...Quantification of comet assays. Results are the average of three independent experiments. At least 100 cells were analyzed per sample. Errors are SEM; * = p...damage repair using neutral comet assay. The neutral comet assay (single-cell gel electrophoresis) is a sensitive and rapid technique for quantifying

  16. Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells.

    PubMed

    El-Awady, Raafat A; Semreen, Mohammad H; Saber-Ayad, Maha M; Cyprian, Farhan; Menon, Varsha; Al-Tel, Taleb H

    2016-01-01

    DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.

  17. A novel DNA damage response mediated by DNA mismatch repair in Caenorhabditis elegans: induction of programmed autophagic cell death in non-dividing cells

    PubMed Central

    Moriwaki, Takahito; Kato, Yuichi; Nakamura, Chihiro; Ishikawa, Satoru; Zhang-Akiyama, Qiu-Mei

    2015-01-01

    DNA mismatch repair (MMR) contributes to genome integrity by correcting errors of DNA polymerase and inducing cell death in response to DNA damage. Dysfunction of MMR results in increased mutation frequency and cancer risk. Clinical researches revealed that MMR abnormalities induce cancers of non-dividing tissues, such as kidney and liver. However, how MMR suppresses cancer in non-dividing tissues is not understood. To address that mechanism, we analyzed the roles of MMR in non-dividing cells using Caenorhabditis elegans (C. elegans), in which all somatic cells are non-dividing in the adult stage. In this study, we used stable MMR-mutant lines with a balancer chromosome. First, we confirmed that deficiency of MMR leads to resistance to various mutagens in C. elegans dividing cells. Next, we performed drug resistance assays, and found that MMR-deficient adult worms were resistant to SN1-type alkylating and oxidizing agents. In addition, dead cell staining and reporter assays of an autophagy-related gene demonstrated that the cell death was autophagic cell death. Interestingly, this autophagic cell death was not suppressed by caffeine, implying that MMR induces death of non-dividing cells in an atl-1-independent manner. Hence, we propose the hypothesis that MMR prevents cancers in non-dividing tissues by directly inducing cell death. PMID:26413217

  18. Chromatin structure and DNA damage

    SciTech Connect

    Gale, J.M.

    1987-01-01

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.

  19. Oxidative Stress, Inflammation, and DNA Damage Responses Elicited by Silver, Titanium Dioxide, and Cerium Oxide Nanomaterials

    EPA Science Inventory

    Previous literature on the biological effects of engineered nanomaterials has focused largely on oxidative stress and inflammation endpoints without further investigating potential pathways. Here we examine time-sensitive biological response pathways affected by engineered nanoma...

  20. TH-C-18A-09: Exam and Patient Parameters Affecting the DNA Damage Response Following CT Studies

    SciTech Connect

    Elgart, S; Adibi, A; Bostani, M; Ruehm, S; Enzmann, D; McNitt-Gray, M; Iwamoto, K

    2014-06-15

    Purpose: To identify exam and patient parameters affecting the biological response to CT studies using in vivo and ex vivo blood samples. Methods: Blood samples were collected under IRB approval from 16 patients undergoing clinically-indicated CT exams. Blood was procured prior to, immediately after and 30minutes following irradiation. A sample of preexam blood was placed on the patient within the exam region for ex vivo analysis. Whole blood samples were fixed immediately following collection and stained for γH2AX to assess DNA damage response (DDR). Median fluorescence of treated samples was compared to non-irradiated control samples for each patient. Patients were characterized by observed biological kinetic response: (a) fast — phosphorylation increased by 2minutes and fell by 30minutes, (b) slow — phosphorylation continued to increase to 30minutes and (c) none — little change was observed or irradiated samples fell below controls. Total dose values were normalized to exam time for an averaged dose-rate in dose/sec for each exam. Relationships between patient biological responses and patient and exam parameters were investigated. Results: A clearer dose response at 30minutes is observed for young patients (<61yoa; R2>0.5) compared to old patients (>61yoa; R{sup 2}<0.11). Fast responding patients were significantly younger than slow responding patients (p<0.05). Unlike in vivo samples, age did not significantly affect the patient response ex vivo. Additionally, fast responding patients received exams with significantly smaller dose-rate than slow responding patients (p<0.05). Conclusion: Age is a significant factor in the biological response suggesting that DDR may be more rapid in a younger population and slower as the population ages. Lack of an agerelated response ex vivo suggests a systemic response to radiation not present when irradiated outside the body. Dose-rate affects the biological response suggesting that patient response may be related to