RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

PubMed

Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih-Ya; Chen, Benjamin P C; Hittelman, Walter N; Wang, Yi

2011-06-24

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

PubMed Central

Wang, Shu-Huei; Lin, Pei-Ya; Chiu, Ya-Chen; Huang, Ju-Sui; Kuo, Yi-Tsen; Wu, Jen-Chine; Chen, Chin-Chuan

2015-01-01

Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity. PMID:26218133

Oxidation in the nucleotide pool, the DNA damage response and cellular senescence: Defective bricks build a defective house.

PubMed

Rai, Priyamvada

2010-11-28

Activation of persistent DNA damage response (DDR) signaling is associated with the induction of a permanent proliferative arrest known as cellular senescence, a phenomenon intrinsically linked to both tissue aging as well as tumor suppression. The DNA damage observed in senescent cells has been attributed to elevated levels of reactive oxygen species (ROS), failing DNA damage repair processes, and/or oncogenic activation. It is not clear how labile molecules such as ROS are able to damage chromatin-bound DNA to a sufficient extent to invoke persistent DNA damage and DDR signaling. Recent evidence suggests that the nucleotide pool is a significant target for oxidants and that oxidized nucleotides, once incorporated into genomic DNA, can lead to the induction of a DNA strand break-associated DDR that triggers senescence in normal cells and in cells sustaining oncogene activation. Evasion of this DDR and resulting senescence is a key step in tumor progression. This review will explore the role of oxidation in the nucleotide pool as a major effector of oxidative stress-induced genotoxic damage and DDR in the context of cellular senescence and tumorigenic transformation. 2010 Elsevier B.V. All rights reserved.

Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.

PubMed

Nilsson, Kersti; Wu, Chengjun; Schwartz, Stefan

2018-06-12

Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

PubMed

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

PubMed Central

Lauretti, Elisabetta; Hulse, Michael; Siciliano, Micheal; Lupey-Green, Lena N.; Abraham, Aaron; Skorski, Tomasz; Tempera, Italo

2018-01-01

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers. PMID:29535829

Effects of Military activity and habitat quality on DNA damage and oxidative stress in the largest population of the Federally threatened gopher tortoise.

PubMed

Theodorakis, Christopher W; Adams, S Marshall; Smith, Chandra; Rotter, Jamie; Hay, Ashley; Eslick, Joy

2017-12-01

Department of Defense lands are essential for providing important habitat for threatened, endangered, and at-risk species (TER-S). However, there is little information on the effects of military-related contaminants on TER-S on these lands in field situations. Thus, this study examined genotoxicity and oxidative stress in gopher tortoises (Gopherus polyphemus) on Camp Shelby, MS-the largest known population of this species, which is listed as an "endangered species" in Mississippi and a "threatened species" by the U.S. government. Blood was collected from tortoises at 19 different sites on the base with different levels of habitat quality (high-quality and low-quality habitat) and military activity (high, low, and no military activity). Oxidative stress was quantified as lipid peroxidation and GSSG/GSH ratios, while DNA damage was determined using flow cytometry. Our results suggest that: (1) for tortoises residing in low-quality habitats, oxidative stress and DNA damage increased with increasing military activity, while in high-quality habitats, oxidative stress and DNA damage decreased with increasing military activity; (2) in the absence of military activity, tortoises in high-quality habitat had higher levels of oxidative stress and DNA damage than those in low-quality habitat, and (3) there were interactions between military activity, habitat quality, and landuse in terms of the amount of observable DNA damage and oxidative stress. In particular, on high-quality habitat, tortoises from areas with high levels of military activity had lower levels of oxidative stress and DNA damage biomarkers than on reference sites. This may represent a compensatory or hormetic response. Conversely, on low-quality habitats, the level of oxidative stress and DNA damage was lower on the reference sites. Thus, tortoises on higher-quality habitats may have a greater capacity for compensatory responses. In terms of management implications, it is suggested that low quality habitats should be a higher priority for remediation, and lower priority for conducting military activities.

PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry.

PubMed

Maréchal, Alexandre; Li, Ju-Mei; Ji, Xiao Ye; Wu, Ching-Shyi; Yazinski, Stephanie A; Nguyen, Hai Dang; Liu, Shizhou; Jiménez, Amanda E; Jin, Jianping; Zou, Lee

2014-01-23

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR. Copyright © 2014 Elsevier Inc. All rights reserved.

Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Hui; Shi, Qiong; Song, Xiufang

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 observedmore » 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.« less

The production and repair of aflatoxin B sub 1 -induced DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leadon, S.A.

To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of aflatoxin B{sub 1} (AFB{sub 1})-induced damage in the nontranscribed, heterochromatic alpha DNA of monkey cells and in the metallothionein genes of human cells. In confluent cells, AFB{sub 1} adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha DNA is severely deficient, however, removal of AFB{sub 1} adducts from alpha DNA is enhanced by small doses of UV. The repair deficiencies are not observed in actively growing cells. We havemore » also shown that there is preferential repair of AFB{sub 1} damage in active genes. AFB{sub 1} damage is efficiently repaired in the active human metallothionein (hMT) genes, but deficiently repaired in inactive hMT genes. 51 refs., 3 tabs.« less

Maintenance of Genome Stability and Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases

DTIC Science & Technology

2008-03-01

Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases PRINCIPAL INVESTIGATOR: Daniel Mordes...Maintenance of Genome Stability and Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases 5b. GRANT NUMBER W81XWH-06-1-0352 5c...shown that this domain of Dpb11 stimulates the kinase activity of wild-type Mec1-Ddc2 yet did not simulate Mec1-ddc2-top. Thus, we have demonstrated

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

PubMed

Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

2017-10-19

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA-damage response during mitosis induces whole-chromosome missegregation.

PubMed

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

2014-11-01

Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.

Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM

PubMed Central

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

2017-01-01

SUMMARY 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. PMID:27939942

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. Copyright © 2017 Elsevier Inc. All rights reserved.

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.

PubMed

Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H

2017-03-01

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway.

PubMed

Das, Ujjal; Manna, Krishnendu; Khan, Amitava; Sinha, Mahuya; Biswas, Sushobhan; Sengupta, Aaveri; Chakraborty, Anindita; Dey, Sanjit

2017-01-01

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro

PubMed Central

Hossain, M. Zulfiquer; Patel, Kalpesh; Kern, Scott E.

2014-01-01

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. PMID:24842839

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro.

PubMed

Hossain, M Zulfiquer; Patel, Kalpesh; Kern, Scott E

2014-08-01

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

PubMed

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

PubMed

Zheng, Huiyuan; Högberg, Johan; Stenius, Ulla

2017-12-07

Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination

PubMed Central

Dubois, Jean-Christophe; Yates, Maïlyn; Gaudreau-Lapierre, Antoine; Clément, Geneviève; Cappadocia, Laurent; Gaudreau, Luc

2017-01-01

Abstract RPA-coated single-stranded DNA (RPA–ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA–ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR. PMID:28666352

TAO kinases mediate activation of p38 in response to DNA damage

PubMed Central

Raman, Malavika; Earnest, Svetlana; Zhang, Kai; Zhao, Yingming; Cobb, Melanie H

2007-01-01

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM. PMID:17396146

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway.

PubMed

Llanos, Susana; Serrano, Manuel

2010-10-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair

PubMed Central

Arango, Daniel; Parihar, Arti; Villamena, Frederick A.; Wang, Liwen; Freitas, Michael A.; Grotewold, Erich; Doseff, Andrea I.

2014-01-01

Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid. PMID:22985621

Preferential mitochondrial DNA injury caused by glucose oxidase as a steady generator of hydrogen peroxide in human fibroblasts.

PubMed

Salazar, J J; Van Houten, B

1997-11-01

To test the hypothesis that mitochondrial DNA (mtDNA) is more prone to reactive oxygen species (ROS) damage than nuclear DNA, a continuous flux of hydrogen peroxide (H2O2) was produced with the glucose/glucose oxidase system. Using a horse radish peroxidase (HRPO)-based colorimetric assay to detect H2O2, glucose oxidase (GO; 12 mU/ml) produced 95 microM of H2O2 in 1 h, whereas only 46 microM of hydrogen peroxide accumulated in the presence of SV40-transformed human fibroblasts ( approximately 1 x 10(6). DNA damage was assessed in the mitochondira and three nuclear regions using a quantitative PCR assay. GO (12 mU/ml) resulted in more damage to the mitochondrial DNA (2.250 +/- 0.045 lesions/10 kb) than in any one of three nuclear targets, which included the non-expressed beta-globin locus (0.436 +/- 0.029 lesions/10 kb); and the active DNA polymerase b gene (0.442 +/- 0.037 lesions/10 kb); and the active hprt gene (0.310 +/- 0.025). Damage to the mtDNA occurred within 15 min of GO treatment, whereas nuclear damage did not appear until after 30 min, and reached a maximum after 60 min. Repair of mitochondrial damage after a 15 min GO (6 mU/ml) treatment was examined. Mitochondria repaired 50% of the damage after 1 h, and by 6 h all the damage was repaired. Higher doses of GO-generated H202, or more extended treatment periods, lead to mitochondrial DNA damage which was not repaired. Mitochondrial function was monitored using the MTT (3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. A 15 min treatment with 6 mU/ml of GO decreased mitochondrial activity to 80% of the control; the activity recovered completely within 1 h after damage. These data show that GO-generated H202 causes acute damage to mtDNA and function, and demonstrate that this organelle is an important site for the cellular toxicity of ROS.

Comparison of antioxidant enzyme activities and DNA damage in chickpea (Cicer arietinum L.) genotypes exposed to vanadium.

PubMed

Imtiaz, Muhammad; Mushtaq, Muhammad Adnan; Rizwan, Muhammad Shahid; Arif, Muhammad Saleem; Yousaf, Balal; Ashraf, Muhammad; Shuanglian, Xiong; Rizwan, Muhammad; Mehmood, Sajid; Tu, Shuxin

2016-10-01

The present study was done to elucidate the effects of vanadium (V) on photosynthetic pigments, membrane damage, antioxidant enzymes, protein, and deoxyribonucleic acid (DNA) integrity in the following chickpea genotypes: C-44 (tolerant) and Balkasar (sensitive). Changes in these parameters were strikingly dependent on levels of V, at 60 and 120 mg V L(-1) induced DNA damage in Balkasar only, while photosynthetic pigments and protein were decreased from 15 to 120 mg V L(-1) and membrane was also damaged. It was shown that photosynthetic pigments and protein production declined from 15 to 120 mg V L(-1) and the membrane was also damaged, while DNA damage was not observed at any level of V stress in C-44. Moreover, the antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were increased in both genotypes of chickpea against V stress; however, more activities were observed in C-44 than Balkasar. The results suggest that DNA damage in sensitive genotypes can be triggered due to exposure of higher vanadium.

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

PubMed

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

2005-04-15

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

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

PubMed

Ogara, María F; Sirkin, Pablo F; Carcagno, Abel L; Marazita, Mariela C; Sonzogni, Silvina V; Ceruti, Julieta M; Cánepa, Eduardo T

2013-01-01

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiran, Shashi; Oddi, Vineesha; Ramakrishna, Gayatri, E-mail: gayatrirama1@gmail.com

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 ofmore » 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 SIRT7 attenuated p38/JNK activation and also p53 response. • Overall, SIRT7 promoted cellular survival in conditions of genomic stress.« less

Antiproliferative, DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: A structure-activity relationship study.

PubMed

Ho, Si-Han Sherman; Sim, Mei-Yi; Yee, Wei-Loong Sherman; Yang, Tianming; Yuen, Shyi-Peng John; Go, Mei-Lin

2015-11-02

The anticancer agent YM155 is widely investigated as a specific survivin suppressant. More recently, YM155 was found to induce DNA damage and this has raised doubts as to whether survivin is its primary target. In an effort to assess the contribution of DNA damage to the anticancer activity of YM155, several analogs were prepared and evaluated for antiproliferative activity on malignant cells, participation in DNA intercalation and free radical generation by redox cycling. The intact positively charged scaffold was found to be essential for antiproliferative activity and intercalation but was less critical for redox cycling where the minimal requirement was a pared down bicyclic quinone. Side chain requirements at the N(1) and N(3) positions of the scaffold were more alike for redox cycling and intercalation than antiproliferative activity, underscoring yet again, the limited structural overlaps for these activities. Furthermore, antiproliferative activities were poorly correlated to DNA intercalation and redox cycling. Potent antiproliferative activity (IC50 9-23 nM), exceeding that of YM155, was found for a minimally substituted methyl analog AB7. Like YM155 and other dioxonaphthoimidazoliums, AB7 was a modest DNA intercalator but with weak redox cycling activity. Thus, the capacity of this scaffold to inflict direct DNA damage leading to cell death may not be significant and YM155 should not be routinely classified as a DNA damaging agent. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Amelioration of oxidative DNA damage in mouse peritoneal macrophages by Hippophae salicifolia due to its proton (H+) donation capability: Ex vivo and in vivo studies

PubMed Central

Chakraborty, Mainak; Karmakar, Indrajit; Haldar, Sagnik; Das, Avratanu; Bala, Asis; Haldar, Pallab Kanti

2016-01-01

Introduction: The present study evaluates the antioxidant effect of methanol extract of Hippophae salicifolia (MEHS) bark with special emphasis on its role on oxidative DNA damage in mouse peritoneal macrophages. Material and Methods: In vitro antioxidant activity was estimated by standard antioxidant assays whereas the antioxidant activity concluded the H+ donating capacity. Mouse erythrocytes’ hemolysis and peritoneal macrophages’ DNA damage were determined spectrophotometrically. In vivo antioxidant activity of MEHS was determined in carbon tetrachloride-induced mice by studying its effect on superoxide anion production in macrophages cells, superoxide dismutase in the cell lysate, DNA damage, lipid peroxidation, and reduces glutathione. Results: The extract showed good in vitro antioxidant activities whereas the inhibitory concentrations values ranged from 5.80 to 106.5 μg/ml. MEHS significantly (P < 0.05) attenuated the oxidative DNA damage. It also attenuated the oxidative conversion of hemoglobin to methemoglobin and elevation of enzymatic and nonenzymatic antioxidant in cells. Conclusion: The result indicates MEHS has good in vitro-in vivo antioxidant property as well as the protective effect on DNA and red blood cell may be due to its H+ donating property. PMID:27413349

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

PubMed

González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

2015-08-11

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

RhoJ Regulates Melanoma Chemoresistance by Suppressing Pathways that Sense DNA Damage

PubMed Central

Ho, Hsiang; Aruri, Jayavani; Kapadia, Rubina; Mehr, Hootan; White, Michael A.; Ganesan, Anand K.

2012-01-01

Melanomas resist conventional chemotherapeutics in part through intrinsic disrespect of apoptotic checkpoint activation. In this study, using an unbiased genome-wide RNAi screen we identified RhoJ and its effector Pak1, as key modulators of melanoma cell sensitivity to DNA damage. We find that RhoJ activates Pak1 in response to drug-induced DNA damage, which then uncouples ATR from its downstream effectors, ultimately resulting in a blunted DNA damage response (DDR). In addition, ATR suppression leads to the decreased phosphorylation of ATF2, and consequent increased expression of the melanocyte survival gene Sox10 resulting in a higher DDR threshold required to engage melanoma cell death. In the setting of normal melanocyte behavior, this regulatory relationship may facilitate appropriate epidermal melanization in response to UV-induced DNA damage. However, pathological pathway activation during oncogenic transformation produces a tumor that is intrinsically resistant to chemotherapy and has the propensity to accumulate additional mutations. These findings identify DNA damage agents and pharmacological inhibitors of RhoJ/PAK1 as novel synergistic agents that can be used to treat melanomas that are resistant to conventional chemotherapies. PMID:22971344

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

PubMed Central

Llanos, Susana; Serrano, Manuel

2013-01-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage. PMID:20935493

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Protective mechanisms of p53-p21-pRb proteins against DNA damage-induced cell death.

PubMed

Garner, Elizabeth; Raj, Kenneth

2008-02-01

There have been innumerate demonstrations of p53's activity as a tumour suppressor protein with the ability to stimulate cell signalling that can lead to cell cycle arrest and cell death in the event of DNA damage. Despite the solid body of evidence to support these properties of p53, reports have emerged that suggest a role for p53 in protecting cells from cell death. Our recent report highlighted a mechanism by which p53 activity can promote cell survival in the event of DNA damage. Here we present the various mechanisms that are activated by p53 signalling that can confer protection to cells with damaged DNA and emphasise the practical and clinical implications of a more balanced and context-dependent understanding of p53's pro-apoptotic and pro-survival activities.

The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

PubMed

Agarwal, Poonam; Miller, Kyle M

2016-10-01

DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response

PubMed Central

Wang, Yucai; Han, Xiao; Wu, Fangming; Leung, Justin W; Lowery, Megan G; Do, Huong; Chen, Junjie; Shi, Chaowei; Tian, Changlin; Li, Lei; Gong, Weimin

2013-01-01

The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation. PMID:23999858

Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response.

PubMed

Luo, Man; Bao, Zhengqiang; Xu, Feng; Wang, Xiaohui; Li, Fei; Li, Wen; Chen, Zhihua; Ying, Songmin; Shen, Huahao

2018-04-14

The inflammatory cascade can be initiated with the recognition of damaged DNA. Macrophages play an essential role in particulate matter (PM)-induced airway inflammation. In this study, we aim to explore the PM induced DNA damage response of macrophages and its function in airway inflammation. The DNA damage response and inflammatory response were assessed using bone marrow-derived macrophages following PM treatment and mouse model instilled intratracheally with PM. We found that PM induced significant DNA damage both in vitro and in vivo and simultaneously triggered a rapid DNA damage response, represented by nuclear RPA, 53BP1 and γH2AX foci formation. Genetic ablation or chemical inhibition of the DNA damage response sensor amplified the production of cytokines including Cxcl1, Cxcl2 and Ifn-γ after PM stimulation in bone marrow-derived macrophages. Similar to that seen in vitro , mice with myeloid-specific deletion of RAD50 showed higher levels of airway inflammation in response to the PM challenge, suggesting a protective role of DNA damage sensor during inflammation. These data demonstrate that PM exposure induces DNA damage and activation of DNA damage response sensor MRN complex in macrophages. Disruption of MRN complex lead to persistent, unrepaired DNA damage that causes elevated inflammatory response.

ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.

PubMed

You, Zhongsheng; Chahwan, Charly; Bailis, Julie; Hunter, Tony; Russell, Paul

2005-07-01

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.

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

ssDNA damage dependence from singlet oxygen concentration at photodynamic interaction

NASA Astrophysics Data System (ADS)

Klimenko, V. V.; Kaydanov, N. E.; Emelyanov, A. K.; Bogdanov, A. A.

2017-11-01

Single stranded DNA damage at photodynamic treatment with Radachlorin photosensitizer was investigated. Chemical trap method was used to evaluate generation of singlet oxygen in water solution. Interaction of singlet oxygen with ssDNA resulted into decrease of the replication activity of ssDNA. DNA stopped replicating during PCR at irradiation doses greater than 15 J/cm2 and concentration of photosensitizer [PS] = 3.8 μM. The dependence of replication activity of ssDNA on generated singlet oxygen concentration was identified.

USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair.

PubMed

Khoronenkova, Svetlana V; Dianov, Grigory L

2013-02-01

The E3 ubiquitin ligase Mule/ARF-BP1 plays an important role in the cellular DNA damage response by controlling base excision repair and p53 protein levels. However, how the activity of Mule is regulated in response to DNA damage is currently unknown. Here, we report that the Ser18-containing isoform of the USP7 deubiquitylation enzyme (USP7S) controls Mule stability by preventing its self-ubiquitylation and subsequent proteasomal degradation. We find that in response to DNA damage, downregulation of USP7S leads to self-ubiquitylation and proteasomal degradation of Mule, which eventually leads to p53 accumulation. Cells that are unable to downregulate Mule show reduced ability to upregulate p53 levels in response to DNA damage. We also find that, as Mule inactivation is required for stabilization of base excision repair enzymes, the failure of cells to downregulate Mule after DNA damage results in deficient DNA repair. Our data describe a novel mechanism by which Mule is regulated in response to DNA damage and coordinates cellular DNA damage responses and DNA repair.

In vitro antioxidant properties, DNA damage protective activity, and xanthine oxidase inhibitory effect of cajaninstilbene acid, a stilbene compound derived from pigeon pea [Cajanus cajan (L.) Millsp.] leaves.

PubMed

Wu, Nan; Kong, Yu; Fu, Yujie; Zu, Yuangang; Yang, Zhiwei; Yang, Mei; Peng, Xiao; Efferth, Thomas

2011-01-12

The antioxidant properties, DNA damage protective activities, and xanthine oxidase (XOD) inhibitory effect of cajaninstilbene acid (CSA) derived from pigeon pea leaves were studied in the present work. Compared with resveratrol, CSA showed stronger antioxidant properties, DNA damage protective activity, and XOD inhibition activity. The IC(50) values of CSA for superoxide radical scavenging, hydroxyl radical scavenging, nitric oxide scavenging, reducing power, lipid peroxidation, and XOD inhibition were 19.03, 6.36, 39.65, 20.41, 20.58, and 3.62 μM, respectively. CSA possessed good protective activity from oxidative DNA damage. Furthermore, molecular docking indicated that CSA was more potent than resveratrol or allopurinol to interact with the active site of XOD (calculated free binding energy: -229.71 kcal mol(-1)). On the basis of the results, we conclude that CSA represents a valuable natural antioxidant source and may potentially be applicable in health food industry.

Bioactive components, antioxidant and DNA damage inhibitory activities of honeys from arid regions.

PubMed

Habib, Hosam M; Al Meqbali, Fatima T; Kamal, Hina; Souka, Usama D; Ibrahim, Wissam H

2014-06-15

Honey serves as a good source of natural antioxidants, which are effective in reducing the risk of occurrence of several diseases. This study was undertaken to address the limited knowledge regarding the polyphenolic content, antioxidant and DNA damage inhibitory activities of honeys produced in arid regions and compare them with well-recognized honeys from non-arid regions. Different types of honey were assessed for their contents of total phenolics, total flavonoids, and certain types of phenolic compounds. The antioxidant capacity of honey was evaluated by ferric-reducing/antioxidant power assay (FRAP), free radical-scavenging activity (DPPH), nitric oxide (NO) radical-scavenging assay, total antioxidant activity, and DNA damage. Results clearly showed significant differences among honeys with all the evaluated parameters. Results also showed that one or more types of honey from arid regions contained higher levels of phenolic compounds, free radical-scavenging activities, or DNA damage inhibitory activities compared with the evaluated honeys from non-arid regions. Copyright © 2013 Elsevier Ltd. All rights reserved.

The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

PubMed Central

Hamperl, Stephan; Cimprich, Karlene A.

2014-01-01

Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. PMID:24746923

Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasegawa, Tatsuya, E-mail: tatsuya.hasegawa@to.shiseido.co.jp; Nakashima, Masaya; Suzuki, Yoshiharu

Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE{sub 2}. In addition, inhibition of DNA damage repair by knockdown of XPA,more » which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. - Highlights: • UVB radiation induces NLRP3 inflammasome activation in human keratinocytes. • NLRP3 knockdown suppresses production of UVB-induced inflammatory mediators. • UVB-induced DNA damage triggers NLRP3 inflammasome activation. • NLRP3 expression in human epidermis is elevated in response to UV radiation.« less

Organic honey supplementation reverses pesticide-induced genotoxicity by modulating DNA damage response.

PubMed

Alleva, Renata; Manzella, Nicola; Gaetani, Simona; Ciarapica, Veronica; Bracci, Massimo; Caboni, Maria Fiorenza; Pasini, Federica; Monaco, Federica; Amati, Monica; Borghi, Battista; Tomasetti, Marco

2016-10-01

Glyphosate (GLY) and organophosphorus insecticides such as chlorpyrifos (CPF) may cause DNA damage and cancer in exposed individuals through mitochondrial dysfunction. Polyphenols ubiquitously present in fruits and vegetables, have been viewed as antioxidant molecules, but also influence mitochondrial homeostasis. Here, honey containing polyphenol compounds was evaluated for its potential protective effect on pesticide-induced genotoxicity. Honey extracts from four floral organic sources were evaluated for their polyphenol content, antioxidant activity, and potential protective effects on pesticide-related mitochondrial destabilization, reactive oxygen and nitrogen species formation, and DNA damage response in human bronchial epithelial and neuronal cells. The protective effect of honey was, then evaluated in a residential population chronically exposed to pesticides. The four honey types showed a different polyphenol profile associated with a different antioxidant power. The pesticide-induced mitochondrial dysfunction parallels ROS formation from mitochondria (mtROS) and consequent DNA damage. Honey extracts efficiently inhibited pesticide-induced mtROS formation, and reduced DNA damage by upregulation of DNA repair through NFR2. Honey supplementation enhanced DNA repair activity in a residential population chronically exposed to pesticides, which resulted in a marked reduction of pesticide-induced DNA lesions. These results provide new insight regarding the effect of honey containing polyphenols on pesticide-induced DNA damage response. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model.

PubMed

Kelner, Michael J; McMorris, Trevor C; Rojas, Rafael J; Estes, Leita A; Suthipinijtham, Pharnuk

2008-12-01

Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko

2014-09-26

Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the processmore » by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.« less

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

PubMed

Neves-Costa, Ana; Moita, Luis F

2017-03-01

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. © 2016 Federation of European Biochemical Societies.

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c

PubMed Central

González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A.

2015-01-01

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity. PMID:26216969

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.

Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

PubMed Central

Friedman, Joshua I.; Stivers, James T.

2010-01-01

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

PubMed

Culyba, Matthew J; Kubiak, Jeffrey M; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2018-06-01

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

Microbial pathogens trigger host DNA double-strand breaks whose abundance is reduced by plant defense responses.

PubMed

Song, Junqi; Bent, Andrew F

2014-04-01

Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs) in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS) is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues.

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts

PubMed Central

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-01-01

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention. PMID:26447614

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.

PubMed

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-11-24

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention.

The ovarian DNA damage repair response is induced prior to phosphoramide mustard-induced follicle depletion, and ataxia telangiectasia mutated inhibition prevents PM-induced follicle depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fishermore » 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96 h in medium containing DMSO ± 60 μM PM or KU 55933 (48 h; 10 nM). PM-induced activation of DNA damage repair genes was observed as early as 12 h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96 h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity. - Highlights: • PM exposure induces DNA damage repair gene expression. • Inhibition of ATM prevented PM-induced follicle depletion. • PKCδ deficiency did not impact PM-induced ovotoxicity.« less

HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

PubMed Central

Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

2008-01-01

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

Chromatin Relaxation-Mediated Induction of p19INK4d Increases the Ability of Cells to Repair Damaged DNA

PubMed Central

Carcagno, Abel L.; Marazita, Mariela C.; Sonzogni, Silvina V.; Ceruti, Julieta M.; Cánepa, Eduardo T.

2013-01-01

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies. PMID:23593412

Cisplatin: mode of cytotoxic action and molecular basis of resistance.

PubMed

Siddik, Zahid H

2003-10-20

Cisplatin is one of the most potent antitumor agents known, displaying clinical activity against a wide variety of solid tumors. Its cytotoxic mode of action is mediated by its interaction with DNA to form DNA adducts, primarily intrastrand crosslink adducts, which activate several signal transduction pathways, including those involving ATR, p53, p73, and MAPK, and culminate in the activation of apoptosis. DNA damage-mediated apoptotic signals, however, can be attenuated, and the resistance that ensues is a major limitation of cisplatin-based chemotherapy. The mechanisms responsible for cisplatin resistance are several, and contribute to the multifactorial nature of the problem. Resistance mechanisms that limit the extent of DNA damage include reduced drug uptake, increased drug inactivation, and increased DNA adduct repair. Origins of these pharmacologic-based mechanisms, however, are at the molecular level. Mechanisms that inhibit propagation of the DNA damage signal to the apoptotic machinery include loss of damage recognition, overexpression of HER-2/neu, activation of the PI3-K/Akt (also known as PI3-K/PKB) pathway, loss of p53 function, overexpression of antiapoptotic bcl-2, and interference in caspase activation. The molecular signature defining the resistant phenotype varies between tumors, and the number of resistance mechanisms activated in response to selection pressures dictates the overall extent of cisplatin resistance.

Antioxidant and DNA damage protective properties of anthocyanin-rich extracts from Hibiscus and Ocimum: a comparative study.

PubMed

Sarkar, Biswatrish; Kumar, Dhananjay; Sasmal, Dinakar; Mukhopadhyay, Kunal

2014-01-01

Anthocyanin extracts (AEs) from Ocimum tenuiflorum (leaf), Hibiscus rosa-sinensis (petal) and Hibiscus sabdariffa (calyx) were investigated and compared for in vitro antioxidant activity and DNA damage protective property. Total phenolic content (TPC) and total anthocyanin content (TAC) of the AEs were determined and the major anthocyanins were characterised. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical-scavenging activity, 2-deoxy-D-ribose degradation assay and lipid peroxidation assay. The protective property of the AEs was also examined against oxidative DNA damage by H2O2 and UV using pUC19 plasmid. All the AEs particularly those from O. tenuiflorum demonstrated efficient antioxidant activity and protected DNA from damage. Strong correlation between antioxidant capacity and TPC and TAC was observed. Significant correlation between antioxidant capacity and TPC and TAC ascertained that phenolics and anthocyanins were the major contributors of antioxidant activity.

Ultrasonic irradiation enhanced the ability of Fluorescein-DA-Fe(III) on sonodynamic and sonocatalytic damages of DNA molecules.

PubMed

Wu, Qiong; Chen, Xia; Jia, Lizhen; Wang, Yi; Sun, Ying; Huang, Xingjun; Shen, Yuxiang; Wang, Jun

2017-11-01

The interaction of DNA with Bis [N,N-bis (carboxymethyl) aminomethyl] fluorescein-Ferrous(III) (Fluorescein-DA-Fe(III)) with dual functional (sonodynamic and sonocatalytic) activity was studied by UV-vis spectroscopy, fluorescence spectroscopy, FT-IR spectroscopy, circular dichroism (CD) spectroscopy and viscosity measurements. And then, the damage of DNA caused by Fluorescein-DA-Fe(III) under ultrasonic irradiation (US) was researched by agarose gel electrophoresis and cytotoxicity assay. Meanwhile, some influenced factors such as ultrasonic irradiation time and Fluorescein-DA-Fe(III) concentration on the damage degree of DNA molecules were also examined. As a control, for Bis [N,N-bis (carboxymethyl) aminomethyl] fluorescein (Fluorescein-DA), the same experiments were carried out. The results showed that both Fluorescein-DA-Fe(III) and Fluorescein-DA can interact with DNA molecules. Under ultrasonic irradiation, Fluorescein-DA shows sonodynamic activity, which can damage DNA molecules. While, in the presence of Fe(III) ion, the Fluorescein-DA-Fe(III) displays not only sonodynamic activity but also sonocatalytic activity under ultrasonic irradiation, which injures DNA more serious than Fluorescein-DA. The researches confirmed the dual function (sonodynamic activity and sonocatalytic activity) of Fluorescein-DA-Fe(III) and expanded the usage of Fluorescein-DA-Fe(III) as a sonosensitizer in sonodynamic therapy (SDT). Copyright © 2017 Elsevier B.V. All rights reserved.

Black soybean seed coat polyphenols prevent AAPH-induced oxidative DNA-damage in HepG2 cells

PubMed Central

Yoshioka, Yasukiyo; Li, Xiu; Zhang, Tianshun; Mitani, Takakazu; Yasuda, Michiko; Nanba, Fumio; Toda, Toshiya; Yamashita, Yoko; Ashida, Hitoshi

2017-01-01

Black soybean seed coat extract (BE), which contains abundant polyphenols such as procyanidins, cyanidin 3-glucoside, (+)-catechin, and (−)­epicatechin, has been reported on health beneficial functions such as antioxidant activity, anti-inflammatory, anti-obesity, and anti-diabetic activities. In this study, we investigated that prevention of BE and its polyphenols on 2,2'-azobis(2-methylpropionamide) dihydrochloride (AAPH)-induced oxidative DNA damage, and found that these polyphenols inhibited AAPH-induced formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for oxidative DNA damage in HepG2 cells. Under the same conditions, these polyphenols also inhibited AAPH-induced accumulation of reactive oxygen species (ROS) in the cells. Inhibition of ROS accumulation was observed in both cytosol and nucleus. It was confirmed that these polyphenols inhibited formation of AAPH radical using oxygen radical absorbance capacity assay under the cell-free conditions. These results indicate that polyphenols in BE inhibit free radical-induced oxidative DNA damages by their potent antioxidant activity. Thus, BE is an effective food material for prevention of oxidative stress and oxidative DNA damages. PMID:28366989

Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex class I-related chain A and B in cancer cells.

PubMed

Nakajima, Nakako Izumi; Niimi, Atsuko; Isono, Mayu; Oike, Takahiro; Sato, Hiro; Nakano, Takashi; Shibata, Atsushi

2017-08-01

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low‑dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage‑dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage‑dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

NASA Astrophysics Data System (ADS)

Ramesh, Govindarajan; Wu, Honglu

Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability

PubMed Central

Mirza, Sameer; Katafiasz, Bryan J.; Kumar, Rakesh; Wang, Jun; Mohibi, Shakur; Jain, Smrati; Gurumurthy, Channabasavaiah Basavaraju; Pandita, Tej K.; Dave, Bhavana J.; Band, Hamid; Band, Vimla

2012-01-01

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3−/− cells. Notably, Ada3−/− cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G₁ or G₂ phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3fl/fl and Ada3−/− cells confirmed higher residual DNA damage in Ada3−/− cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability. PMID:23095635

A critical role for topoisomerase IIb and DNA double strand breaks in transcription

PubMed Central

Calderwood, Stuart K.

2016-01-01

ABSTRACT Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

PubMed

Calderwood, Stuart K

2016-05-26

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

PubMed

Douthwright, Stephen; Sluder, Greenfield

2014-10-01

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. © 2014 Wiley Periodicals, Inc.

DNA damage in children with scoliosis following X-ray exposure.

PubMed

Himmetoglu, S; Guven, M F; Bilsel, N; Dincer, Y

2014-10-14

It has been suggested that cancer incidence is high in subjects with scoliosis who are relatively more often exposed to X--ray for diagnosis and follow--up. X--ray is a kind of ionizing radiation and leads to formation of oxygen free radicals which are capable of damage to DNA, thus altered gen expression and mutation. p53 tumor suppressor gene plays a crucial role in the damage response. It controls the checkpoint of cell cycle and redirects the cell metabolism to either repair of damaged DNA or apoptosis as response to DNA damage. The aim of the present study was to examine serum levels of 8--Hydroxydeoxyguanosine (8--OHdG), a strongly mutagenic product of oxidative DNA damage, p53, superoxide dismutase (SOD) and glutathione peroxidase (G--Px), as antioxidant activity, in children with scoliosis who had got whole spine radiograph two times during the last year. A total of 31 children with adolescent idiopathic scoliosis and age--matched 21 healthy children were included in the study. Serum levels of 8--OHdG and p53 were measured with ELISA kits. SOD and G--Px activities were determined with spectrophotometric assays. Serum levels of 8--OHdG and p53 were found to be higher (P<0.001 and P<0.01, respectively), SOD activity was found to be lower (P<0.001) in the children with scoliosis as compared to age--matched controls. There was no significant difference between the groups for G--Px activity. Our data show that X--ray exposure causes increased 8--OHdG level, and decreased SOD activity, which both may reflect a tumor promoting condition. Increased p53 level may be interpreted as a compensatory effort of cell to X--ray mediated DNA damage.

DNA damage in children with scoliosis following X-ray exposure.

PubMed

Himmetoglu, S; Guven, M F; Bilsel, N; Dincer, Y

2015-06-01

It has been suggested that cancer incidence is high in subjects with scoliosis who are relatively more often exposed to X-ray for diagnosis and follow-up. X-ray is a kind of ionizing radiation and leads to formation of oxygen free radicals which are capable of damage to DNA, thus altered gen expression and mutation. p53 tumor suppressor gene plays a crucial role in the damage response. It controls the checkpoint of cell cycle and redirects the cell metabolism to either repair of damaged DNA or apoptosis as response to DNA damage. The aim of the present study was to examine serum levels of 8-Hydroxydeoxyguanosine (8-OHdG), a strongly mutagenic product of oxidative DNA damage, p53, superoxide dismutase (SOD) and glutathione peroxidase (G-Px), as antioxidant activity, in children with scoliosis who had got whole spine radiograph two times during the last year. A total of 31 children with adolescent idiopathic scoliosis and 21 age-matched healthy children were included in the study. Serum levels of 8-OHdG and p53 were measured with ELISA kits. SOD and G-Px activities were determined with spectrophotometric assays. Serum levels of 8-OHdG and p53 were found to be higher (P<0.001 and P<0.01, respectively), SOD activity was found to be lower (P<0.001) in the children with scoliosis as compared to age-matched controls. There was no significant difference between the groups for G-Px activity. Our data show that X-ray exposure causes increased 8-OHdG level, and decreased SOD activity, which both may reflect a tumor promoting condition. Increased p53 level may be interpreted as a compensatory effort of cell to X-ray mediated DNA damage.

Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA.

PubMed

Guzder, S N; Sung, P; Prakash, L; Prakash, S

1998-11-20

Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes.

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; < 10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein.more » Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. - Highlights: • Low micromolar concentration of uranium inhibits polymerase-1 (PARP-1) activity. • Uranium causes zinc loss from multiple DNA repair proteins. • Uranium enhances retention of DNA damage caused by ultraviolet radiation. • Zinc reverses the effects of uranium on PARP activity and DNA damage repair.« less

Antioxidant and prooxidant effects of polyphenol compounds on copper-mediated DNA damage.

PubMed

Perron, Nathan R; García, Carla R; Pinzón, Julio R; Chaur, Manuel N; Brumaghim, Julia L

2011-05-01

Inhibition of copper-mediated DNA damage has been determined for several polyphenol compounds. The 50% inhibition concentration values (IC(50)) for most of the tested polyphenols are between 8 and 480 μM for copper-mediated DNA damage prevention. Although most tested polyphenols were antioxidants under these conditions, they generally inhibited Cu(I)-mediated DNA damage less effectively than Fe(II)-mediated damage, and some polyphenols also displayed prooxidant activity. Because semiquinone radicals and hydroxyl radical adducts were detected by EPR spectroscopy in solutions of polyphenols, Cu(I), and H(2)O(2), it is likely that weak polyphenol-Cu(I) interactions permit a redox-cycling mechanism, whereby the necessary reactants to cause DNA damage (Cu(I), H(2)O(2), and reducing agents) are regenerated. The polyphenol compounds that prevent copper-mediated DNA damage likely follow a radical scavenging pathway as determined by EPR spectroscopy. Copyright © 2011 Elsevier Inc. All rights reserved.

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis[OPEN

PubMed Central

Liu, Chun-Hsin; Finke, Andreas; Díaz, Mariana; Rozhon, Wilfried; Poppenberger, Brigitte; Baubec, Tuncay; Pecinka, Ales

2015-01-01

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway. PMID:26023162

Exploiting Tumor-Activated Testes Proteins to Enhance Efficacy of First-Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2015-10-01

TERMS Cancer Testis Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF...Cancer Testis Antigen (CTA) o Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What

Molecular insights into the recruitment of TFIIH to sites of DNA damage

PubMed Central

Oksenych, Valentyn; de Jesus, Bruno Bernardes; Zhovmer, Alexander; Egly, Jean-Marc; Coin, Frédéric

2009-01-01

XPB and XPD subunits of TFIIH are central genome caretakers involved in nucleotide excision repair (NER), although their respective role within this DNA repair pathway remains difficult to delineate. To obtain insight into the function of XPB and XPD, we studied cell lines expressing XPB or XPD ATPase-deficient complexes. We show the involvement of XPB, but not XPD, in the accumulation of TFIIH to sites of DNA damage. Recruitment of TFIIH occurs independently of the helicase activity of XPB, but requires two recently identified motifs, a R-E-D residue loop and a Thumb-like domain. Furthermore, we show that these motifs are specifically involved in the DNA-induced stimulation of the ATPase activity of XPB. Together, our data demonstrate that the recruitment of TFIIH to sites of damage is an active process, under the control of the ATPase motifs of XPB and suggest that this subunit functions as an ATP-driven hook to stabilize the binding of the TFIIH to damaged DNA. PMID:19713942

Genomic biomarkers and clinical outcomes of physical activity.

PubMed

Izzotti, Alberto

2011-07-01

Clinical and experimental studies in humans provide evidence that moderate physical activity significantly decreases artery oxidative damage to nuclear DNA, DNA-adducts related to age and dyslipedemia, and mitochondrial DNA damage. Maintenance of adequate mitochondrial function is crucial for preventing lipid accumulation and peroxidation occurring in atherosclerosis. Studies performed on human muscle biopsies analyzing gene expression in living humans reveal that physically active subjects improve the expression of genes involved in mitochondrial function and of related microRNAs. The attenuation of oxidative damage to nuclear and mitochondrial DNA by physical activity resulted in beneficial effects due to polymorphisms of glutathione S-transferases genes. Subjects bearing null GSTM1/T1 polymorphisms have poor life expectancy in the case of being sedentary, which was increased 2.6-fold in case they performed physical activity. These findings indicate that the preventive effect of physical activity undergoes interindividual variation affected by genetic polymorphisms. © 2011 New York Academy of Sciences.

Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress.

PubMed

Cheng, Ni; Wang, Yuan; Gao, Hui; Yuan, Jialing; Feng, Fan; Cao, Wei; Zheng, Jianbin

2013-09-01

The protective effect of extract of Crataegus pinnatifida (Rosaceae) pollen (ECPP) on the DNA damage response to oxidative stress was investigated and assessed with an alkaline single-cell gel electrophoresis (SCGE) assay and pBR322 plasmid DNA breaks in site-specific and non-site-specific systems. Total phenolic content, total flavonoid content, individual phenolic compounds, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavenging activity, FRAP, and chelating activity) were also determined. The results showed that ECPP possessed a strong ability to protect DNA from being damaged by hydroxyl radicals in both the site-specific system and the non-site-specific system. It also exhibited a cytoprotection effect in mouse lymphocytes against H₂O₂-induced DNA damage. These protective effects may be related to its high total phenolic content (17.65±0.97 mg GAE/g), total flavonoid content (8.04±0.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.48±0.21 mg Na₂EDTA/g). Copyright © 2013 Elsevier Ltd. All rights reserved.

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity

PubMed Central

Im, Michelle; Dagnino, Lina

2018-01-01

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity. PMID:29568383

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity.

PubMed

Im, Michelle; Dagnino, Lina

2018-03-02

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity.

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts.

PubMed

Nesnow, Stephen; Davis, Christine; Nelson, Garret B; Lambert, Guy; Padgett, William; Pimentel, Maria; Tennant, Alan H; Kligerman, Andrew D; Ross, Jeffrey A

2002-11-26

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.

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. © 2015 Takada et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.

PubMed

Smith, Rebecca; Sellou, Hafida; Chapuis, Catherine; Huet, Sébastien; Timinszky, Gyula

2018-05-04

One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

PubMed

Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

2017-09-15

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

PubMed

She, Hua; Mao, Zixu

2017-01-01

The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

Involvement of oxidatively damaged DNA and repair in cancer development and aging

PubMed Central

Tudek, Barbara; Winczura, Alicja; Janik, Justyna; Siomek, Agnieszka; Foksinski, Marek; Oliński, Ryszard

2010-01-01

DNA damage and DNA repair may mediate several cellular processes, like replication and transcription, mutagenesis and apoptosis and thus may be important factors in the development and pathology of an organism, including cancer. DNA is constantly damaged by reactive oxygen species (ROS) and reactive nitrogen species (RNS) directly and also by products of lipid peroxidation (LPO), which form exocyclic adducts to DNA bases. A wide variety of oxidatively-generated DNA lesions are present in living cells. 8-oxoguanine (8-oxoGua) is one of the best known DNA lesions due to its mutagenic properties. Among LPO-derived DNA base modifications the most intensively studied are ethenoadenine and ethenocytosine, highly miscoding DNA lesions considered as markers of oxidative stress and promutagenic DNA damage. Although at present it is impossible to directly answer the question concerning involvement of oxidatively damaged DNA in cancer etiology, it is likely that oxidatively modified DNA bases may serve as a source of mutations that initiate carcinogenesis and are involved in aging (i.e. they may be causal factors responsible for these processes). To counteract the deleterious effect of oxidatively damaged DNA, all organisms have developed several DNA repair mechanisms. The efficiency of oxidatively damaged DNA repair was frequently found to be decreased in cancer patients. The present work reviews the basis for the biological significance of DNA damage, particularly effects of 8-oxoGua and ethenoadduct occurrence in DNA in the aspect of cancer development, drawing attention to the multiplicity of proteins with repair activities. PMID:20589166

Opposing effects of pericentrin and microcephalin on the pericentriolar material regulate CHK1 activation in the DNA damage response.

PubMed

Antonczak, A K; Mullee, L I; Wang, Y; Comartin, D; Inoue, T; Pelletier, L; Morrison, C G

2016-04-14

Genotoxic stresses lead to centrosome amplification, a frequently-observed feature in cancer that may contribute to genome instability and to tumour cell invasion. Here we have explored how the centrosome controls DNA damage responses. For most of the cell cycle, centrosomes consist of two centrioles embedded in the proteinaceous pericentriolar material (PCM). Recent data indicate that the PCM is not an amorphous assembly of proteins, but actually a highly organised scaffold around the centrioles. The large coiled-coil protein, pericentrin, participates in PCM assembly and has been implicated in the control of DNA damage responses (DDRs) through its interactions with checkpoint kinase 1 (CHK1) and microcephalin (MCPH1). CHK1 is required for DNA damage-induced centrosome amplification, whereas MCPH1 deficiency greatly increases the amplification seen after DNA damage. We found that the PCM showed a marked expansion in volume and a noticeable change in higher-order organisation after ionising radiation treatment. PCM expansion was dependent on CHK1 kinase activity and was potentiated by MCPH1 deficiency. Furthermore, pericentrin deficiency or mutation of a separase cleavage site blocked DNA damage-induced PCM expansion. The extent of nuclear CHK1 activation after DNA damage reflected the level of PCM expansion, with a reduction in pericentrin-deficient or separase cleavage site mutant-expressing cells, and an increase in MCPH1-deficient cells that was suppressed by the loss of pericentrin. Deletion of the nuclear export signal of CHK1 led to its hyperphosphorylation after irradiation and reduced centrosome amplification. Deletion of the nuclear localisation signal led to low CHK1 activation and low centrosome amplification. From these data, we propose a feedback loop from the PCM to the nuclear DDR in which CHK1 regulates pericentrin-dependent PCM expansion to control its own activation.

Noncanonical ATM Activation and Signaling in Response to Transcription-Blocking DNA Damage.

PubMed

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

2017-01-01

Environmental genotoxins and metabolic byproducts generate DNA lesions that can cause genomic instability and disrupt tissue homeostasis. To ensure genomic integrity, cells employ mechanisms that convert signals generated by stochastic DNA damage into organized responses, including activation of repair systems, cell cycle checkpoints, and apoptotic mechanisms. DNA damage response (DDR) signaling pathways coordinate these responses and determine cellular fates in part, by transducing signals that modulate RNA metabolism. One of the master DDR coordinators, the Ataxia Telangiectasia Mutated (ATM) kinase, has a fundamental role in mediating DNA damage-induced changes in mRNA synthesis. ATM acts by modulating a variety of RNA metabolic pathways including nascent RNA splicing, a process catalyzed by the spliceosome. Interestingly, ATM and the spliceosome influence each other's activity in a reciprocal manner by a pathway that initiates when transcribing RNA polymerase II (RNAPII) encounters DNA lesions that prohibit forward translocation. In response to stalling of RNAPII assembly of late-stage spliceosomes is disrupted resulting in increased splicing factor mobility. Displacement of spliceosomes from lesion-arrested RNA polymerases facilitates formation of R-loops between the nascent RNA and DNA adjacent to the transcription bubble. R-loops signal for noncanonical ATM activation which in quiescent cells occurs in absence of detectable dsDNA breaks. In turn, activated ATM signals to regulate spliceosome dynamics and AS genome wide.This chapter describes the use of fluorescence microscopy methods that can be used to evaluate noncanonical ATM activation by transcription-blocking DNA damage. First, we present an immunofluorescence-detection method that can be used to evaluate ATM activation by autophosphorylation, in fixed cells. Second, we present a protocol for Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged splicing factors, a highly sensitive and reproducible readout to measure in living cells, the ATM influence on the spliceosome. These approaches have been extensively used in our laboratory for a number of cell lines of various origins and are particularly informative when used in primary cells that can be synchronized in quiescence, to avoid generation of replication stress-induced dsDNA breaks and consequent ATM activation through its canonical pathway.

Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

PubMed Central

Solomon, Jonathan M.; Pasupuleti, Rao; Xu, Lei; McDonagh, Thomas; Curtis, Rory; DiStefano, Peter S.; Huber, L. Julie

2006-01-01

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells. PMID:16354677

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.

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Exploiting Tumor Activated Testes Proteins To Enhance Efficacy of First Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2016-10-01

Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What were the major goals and objectives of

BDNF and exercise enhance neuronal DNA repair by stimulating CREB-mediated production of apurinic/apyrimidinic endonuclease 1.

PubMed

Yang, Jenq-Lin; Lin, Yu-Ting; Chuang, Pei-Chin; Bohr, Vilhelm A; Mattson, Mark P

2014-03-01

Brain-derived neurotrophic factor (BDNF) promotes the survival and growth of neurons during brain development and mediates activity-dependent synaptic plasticity and associated learning and memory in the adult. BDNF levels are reduced in brain regions affected in Alzheimer's, Parkinson's, and Huntington's diseases, and elevation of BDNF levels can ameliorate neuronal dysfunction and degeneration in experimental models of these diseases. Because neurons accumulate oxidative lesions in their DNA during normal activity and in neurodegenerative disorders, we determined whether and how BDNF affects the ability of neurons to cope with oxidative DNA damage. We found that BDNF protects cerebral cortical neurons against oxidative DNA damage-induced death by a mechanism involving enhanced DNA repair. BDNF stimulates DNA repair by activating cyclic AMP response element-binding protein (CREB), which, in turn, induces the expression of apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision DNA repair pathway. Suppression of either APE1 or TrkB by RNA interference abolishes the ability of BDNF to protect neurons against oxidized DNA damage-induced death. The ability of BDNF to activate CREB and upregulate APE1 expression is abolished by shRNA of TrkB as well as inhibitors of TrkB, PI3 kinase, and Akt kinase. Voluntary running wheel exercise significantly increases levels of BDNF, activates CREB, and upregulates APE1 in the cerebral cortex and hippocampus of mice, suggesting a novel mechanism whereby exercise may protect neurons from oxidative DNA damage. Our findings reveal a previously unknown ability of BDNF to enhance DNA repair by inducing the expression of the DNA repair enzyme APE1.

Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

PubMed Central

2015-01-01

The kinetic mechanisms by which DNA polymerases catalyze DNA replication and repair have long been areas of active research. Recently discovered Y-family DNA polymerases catalyze the bypass of damaged DNA bases that would otherwise block replicative DNA polymerases and stall replication forks. Unlike DNA polymerases from the five other families, the Y-family DNA polymerases have flexible, solvent-accessible active sites that are able to tolerate various types of damaged template bases and allow for efficient lesion bypass. Their promiscuous active sites, however, also lead to fidelities that are much lower than those observed for other DNA polymerases and give rise to interesting mechanistic properties. Additionally, the Y-family DNA polymerases have several other unique structural features and undergo a set of conformational changes during substrate binding and catalysis different from those observed for replicative DNA polymerases. In recent years, pre-steady-state kinetic methods have been extensively employed to reveal a wealth of information about the catalytic properties of these fascinating noncanonical DNA polymerases. Here, we review many of the recent findings on the kinetic mechanisms of DNA polymerization with undamaged and damaged DNA substrates by the Y-family DNA polymerases, and the conformational dynamics employed by these error-prone enzymes during catalysis. PMID:24716482

Short and long time effects of low temperature Plasma Activated Media on 3D multicellular tumor spheroids

NASA Astrophysics Data System (ADS)

Judée, Florian; Fongia, Céline; Ducommun, Bernard; Yousfi, Mohammed; Lobjois, Valérie; Merbahi, Nofel

2016-02-01

This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H2O2 plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that and OH* are involved in H2O2 formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or -80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors.

Evaluation of Antioxidant and DNA Damage Protection Activity of the Hydroalcoholic Extract of Desmostachya bipinnata L. Stapf

PubMed Central

Bhimathati, Solomon Sunder Raj

2014-01-01

Desmostachya bipinnata Stapf (Poaceae/Gramineae) is an official drug of ayurvedic pharmacopoeia. Various parts of this plant were used extensively in traditional and folklore medicine to cure various human ailments. The present study was aimed to evaluate the antioxidant and DNA damage protection activity of hydroalcoholic extract of Desmostachya bipinnata both in vitro and in vivo, to provide scientific basis for traditional usage of this plant. The extract showed significant antioxidant activity in a dose-dependent manner with an IC50 value of 264.18 ± 3.47 μg/mL in H2O2 scavenging assay and prevented the oxidative damage to DNA in presence of DNA damaging agent (Fenton's reagent) at a concentration of 50 μg/mL. Also, the presence of extract protected yeast cells in a dose-dependent manner against DNA damaging agent (Hydroxyurea) in spot assay. Moreover, the presence of extract exhibited significant antioxidant activity in vivo by protecting yeast cells against oxidative stressing agent (H2O2). Altogether, the results of current study revealed that Desmostachya bipinnata is a potential source of antioxidants and lends pharmacological credence to the ethnomedical use of this plant in traditional system of medicine, justifying its therapeutic application for free-radical-induced diseases. PMID:24574873

Evaluation of antioxidant and DNA damage protection activity of the hydroalcoholic extract of Desmostachya bipinnata L. Stapf.

PubMed

Golla, Upendarrao; Bhimathati, Solomon Sunder Raj

2014-01-01

Desmostachya bipinnata Stapf (Poaceae/Gramineae) is an official drug of ayurvedic pharmacopoeia. Various parts of this plant were used extensively in traditional and folklore medicine to cure various human ailments. The present study was aimed to evaluate the antioxidant and DNA damage protection activity of hydroalcoholic extract of Desmostachya bipinnata both in vitro and in vivo, to provide scientific basis for traditional usage of this plant. The extract showed significant antioxidant activity in a dose-dependent manner with an IC50 value of 264.18±3.47  μg/mL in H2O2 scavenging assay and prevented the oxidative damage to DNA in presence of DNA damaging agent (Fenton's reagent) at a concentration of 50  μg/mL. Also, the presence of extract protected yeast cells in a dose-dependent manner against DNA damaging agent (Hydroxyurea) in spot assay. Moreover, the presence of extract exhibited significant antioxidant activity in vivo by protecting yeast cells against oxidative stressing agent (H2O2). Altogether, the results of current study revealed that Desmostachya bipinnata is a potential source of antioxidants and lends pharmacological credence to the ethnomedical use of this plant in traditional system of medicine, justifying its therapeutic application for free-radical-induced diseases.

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

PubMed Central

Sasatani, Megumi; Xu, Yanbin; Kawai, Hidehiko; Cao, Lili; Tateishi, Satoshi; Shimura, Tsutomu; Li, Jianxiang; Iizuka, Daisuke; Noda, Asao; Hamasaki, Kanya; Kusunoki, Yoichiro; Kamiya, Kenji

2015-01-01

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure. PMID:25675240

Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

PubMed Central

Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

2013-01-01

Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi.

PubMed

Rojas, Diego A; Urbina, Fabiola; Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo; Maldonado, Edio

2018-02-01

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

Circadian Modulation of 8-Oxoguanine DNA Damage Repair

PubMed Central

Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

2015-01-01

The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

PubMed Central

Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C.M.; Jansen, Jacob G.; Hogenbirk, Marc A.; de Wind, Niels; Jacobs, Heinz

2015-01-01

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (PcnaK164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. PMID:25505145

Differential p53 engagement in response to oxidative and oncogenic stresses in Fanconi anemia mice.

PubMed

Rani, Reena; Li, Jie; Pang, Qishen

2008-12-01

Members of the Fanconi anemia (FA) protein family are involved in repair of genetic damage caused by DNA cross-linkers. It is not clear whether the FA proteins function in oxidative DNA damage and oncogenic stress response. Here, we report that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress. Using a Fanca-/-Trp53-/- double knockout model and a functionally switchable p53 retrovirus, we define the kinetics, dependence, and persistence of p53-mediated response to oxidative and oncogenic stresses in Fanca-/- cells. Notably, oxidative stress induces persistent p53 response in Fanca-/- cells, likely due to accumulation of unrepaired DNA damage. On the other hand, whereas wild-type cells exhibit prolonged response to oncogene activation, the p53-activating signals induced by oncogenic ras are short-lived in Fanca-/- cells, suggesting that Fanca may be required for the cell to engage p53 during constitutive ras activation. We propose that the FA proteins protect cells from stress-induced proliferative arrest and tumor evolution by acting as a modulator of the signaling pathways that link FA to p53.

Differential p53 engagement in response to oxidative and oncogenic stresses in Fanconi anemia mice

PubMed Central

Rani, Reena; Li, Jie; Pang, Qishen

2008-01-01

Members of the Fanconi anemia (FA) protein family are involved in repair of genetic damage caused by DNA cross-linkers. It is not clear whether the FA proteins function in oxidative DNA damage and oncogenic stress response. Here we report that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress. Using a Fanca-/- Trp53-/- double knockout model and a functionally switchable p53 retrovirus, we define the kinetics, dependence, and persistence of p53-mediated response to oxidative and oncogenic stresses in Fanca-/- cells. Notably, oxidative stress induces persistent p53 response in Fanca-/- cells, likely due to accumulation of unrepaired DNA damage. On the other hand, whereas WT cells exhibit prolonged response to oncogene activation, the p53-activating signals induced by oncogenic ras are short-lived in Fanca-/- cells, suggesting that Fanca may be required for the cell to engage p53 during constitutive ras activation. We propose that the FA proteins protect cells from stress-induced proliferative arrest and tumor evolution by acting as a modulator of the signaling pathways that link FA to p53. PMID:19047147

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture

PubMed Central

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. PMID:27821713

Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides.

PubMed

Singh, Satyender; Kumar, Vivek; Vashisht, Kapil; Singh, Priyanka; Banerjee, Basu Dev; Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen; Jain, Sudhir Kumar; Rai, Arvind

2011-11-15

Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p<0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37±2.15 vs. 6.24±1.37 tail% DNA, p<0.001). Further, the workers with CYP2D6*3PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p<0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. Copyright © 2011 Elsevier Inc. All rights reserved.

Protective Effects of Gelam Honey against Oxidative Damage in Young and Aged Rats

PubMed Central

Sahhugi, Zulaikha; Jubri, Zakiah

2014-01-01

Aging is characterized by progressive decline in physiological and body function due to increase in oxidative damage. Gelam honey has been accounted to have high phenolic and nonphenolic content to attenuate oxidative damage. This study was to determine the effect of local gelam honey on oxidative damage of aged rats. Twenty-four male Spraque-Dawley rats were divided into young (2 months) and aged (19 months) groups. Each group was further divided into control (fed with plain water) and supplemented with 2.5 mg/kg body weight of gelam honey for 8 months. DNA damage level was determined by comet assay and plasma malondialdehyde (MDA) by high performance liquid chromatography (HPLC). The activity of blood and cardiac antioxidant enzymes was determined by spectrophotometer. The DNA damage and MDA level were reduced in both gelam honey supplemented groups. Gelam honey increases erythrocytes CAT and cardiac SOD activities in young and cardiac CAT activity in young and aged groups. The DNA damage was increased in the aged group compared to young group, but reduced at the end of the study. The decline of oxidative damage in rats supplemented with gelam honey might be through the modulation of antioxidant enzyme activities. PMID:25505937

S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe

PubMed Central

Lindsay, Howard D.; Griffiths, Dominic J.F.; Edwards, Rhian J.; Christensen, Per U.; Murray, Johanne M.; Osman, Fekret; Walworth, Nancy; Carr, Antony M.

1998-01-01

Checkpoints that respond to DNA structure changes were originally defined by the inability of yeast mutants to prevent mitosis following DNA damage or S-phase arrest. Genetic analysis has subsequently identified subpathways of the DNA structure checkpoints, including the reversible arrest of DNA synthesis. Here, we show that the Cds1 kinase is required to slow S phase in the presence of DNA-damaging agents. Cds1 is phosphorylated and activated by S-phase arrest and activated by DNA damage during S phase, but not during G1 or G2. Activation of Cds1 during S phase is dependent on all six checkpoint Rad proteins, and Cds1 interacts both genetically and physically with Rad26. Unlike its Saccharomyces cerevisiae counterpart Rad53, Cds1 is not required for the mitotic arrest checkpoints and, thus, defines an S-phase specific subpathway of the checkpoint response. We propose a model for the DNA structure checkpoints that offers a new perspective on the function of the DNA structure checkpoint proteins. This model suggests that an intrinsic mechanism linking S phase and mitosis may function independently of the known checkpoint proteins. PMID:9450932

Mediator of DNA damage checkpoint 1 (MDC1) contributes to high NaCl-induced activation of the osmoprotective transcription factor TonEBP/OREBP.

PubMed

Kunin, Margarita; Dmitrieva, Natalia I; Gallazzini, Morgan; Shen, Rong-Fong; Wang, Guanghui; Burg, Maurice B; Ferraris, Joan D

2010-08-11

Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of that factor's transcriptional activity.

Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.

PubMed

Pedroza-García, José-Antonio; Mazubert, Christelle; Del Olmo, Ivan; Bourge, Mickael; Domenichini, Séverine; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; Piñeiro, Manuel; Jarillo, José A; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

2017-03-01

Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis ( Arabidopsis thaliana ). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote. © 2017 American Society of Plant Biologists. All Rights Reserved.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

PubMed

Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

2016-11-29

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

PubMed Central

Hill, Sarah J.; Mordes, Daniel A.; Cameron, Lisa A.; Neuberg, Donna S.; Landini, Serena; Eggan, Kevin; Livingston, David M.

2016-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis. PMID:27849576

In Barrett's esophagus patients and Barrett's cell lines, ursodeoxycholic acid increases antioxidant expression and prevents DNA damage by bile acids.

PubMed

Peng, Sui; Huo, Xiaofang; Rezaei, Davood; Zhang, Qiuyang; Zhang, Xi; Yu, Chunhua; Asanuma, Kiyotaka; Cheng, Edaire; Pham, Thai H; Wang, David H; Chen, Minhu; Souza, Rhonda F; Spechler, Stuart Jon

2014-07-15

Hydrophobic bile acids like deoxycholic acid (DCA), which cause oxidative DNA damage and activate NF-κB in Barrett's metaplasia, might contribute to carcinogenesis in Barrett's esophagus. We have explored mechanisms whereby ursodeoxycholic acid (UDCA, a hydrophilic bile acid) protects against DCA-induced injury in vivo in patients and in vitro using nonneoplastic, telomerase-immortalized Barrett's cell lines. We took biopsies of Barrett's esophagus from 21 patients before and after esophageal perfusion with DCA (250 μM) at baseline and after 8 wk of oral UDCA treatment. DNA damage was assessed by phospho-H2AX expression, neutral CometAssay, and phospho-H2AX nuclear foci formation. Quantitative PCR was performed for antioxidants including catalase and GPX1. Nrf2, catalase, and GPX1 were knocked down with siRNAs. Reporter assays were performed using a plasmid construct containing antioxidant responsive element. In patients, baseline esophageal perfusion with DCA significantly increased phospho-H2AX and phospho-p65 in Barrett's metaplasia. Oral UDCA increased GPX1 and catalase levels in Barrett's metaplasia and prevented DCA perfusion from inducing DNA damage and NF-κB activation. In cells, DCA-induced DNA damage and NF-κB activation was prevented by 24-h pretreatment with UDCA, but not by mixing UDCA with DCA. UDCA activated Nrf2 signaling to increase GPX1 and catalase expression, and protective effects of UDCA pretreatment were blocked by siRNA knockdown of these antioxidants. UDCA increases expression of antioxidants that prevent toxic bile acids from causing DNA damage and NF-κB activation in Barrett's metaplasia. Elucidation of this molecular pathway for UDCA protection provides rationale for clinical trials on UDCA for chemoprevention in Barrett's esophagus. Copyright © 2014 the American Physiological Society.

In Barrett's esophagus patients and Barrett's cell lines, ursodeoxycholic acid increases antioxidant expression and prevents DNA damage by bile acids

PubMed Central

Peng, Sui; Huo, Xiaofang; Rezaei, Davood; Zhang, Qiuyang; Zhang, Xi; Yu, Chunhua; Asanuma, Kiyotaka; Cheng, Edaire; Pham, Thai H.; Wang, David H.; Chen, Minhu; Spechler, Stuart Jon

2014-01-01

Hydrophobic bile acids like deoxycholic acid (DCA), which cause oxidative DNA damage and activate NF-κB in Barrett's metaplasia, might contribute to carcinogenesis in Barrett's esophagus. We have explored mechanisms whereby ursodeoxycholic acid (UDCA, a hydrophilic bile acid) protects against DCA-induced injury in vivo in patients and in vitro using nonneoplastic, telomerase-immortalized Barrett's cell lines. We took biopsies of Barrett's esophagus from 21 patients before and after esophageal perfusion with DCA (250 μM) at baseline and after 8 wk of oral UDCA treatment. DNA damage was assessed by phospho-H2AX expression, neutral CometAssay, and phospho-H2AX nuclear foci formation. Quantitative PCR was performed for antioxidants including catalase and GPX1. Nrf2, catalase, and GPX1 were knocked down with siRNAs. Reporter assays were performed using a plasmid construct containing antioxidant responsive element. In patients, baseline esophageal perfusion with DCA significantly increased phospho-H2AX and phospho-p65 in Barrett's metaplasia. Oral UDCA increased GPX1 and catalase levels in Barrett's metaplasia and prevented DCA perfusion from inducing DNA damage and NF-κB activation. In cells, DCA-induced DNA damage and NF-κB activation was prevented by 24-h pretreatment with UDCA, but not by mixing UDCA with DCA. UDCA activated Nrf2 signaling to increase GPX1 and catalase expression, and protective effects of UDCA pretreatment were blocked by siRNA knockdown of these antioxidants. UDCA increases expression of antioxidants that prevent toxic bile acids from causing DNA damage and NF-κB activation in Barrett's metaplasia. Elucidation of this molecular pathway for UDCA protection provides rationale for clinical trials on UDCA for chemoprevention in Barrett's esophagus. PMID:24852569

Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[α]-pyrene-induced DNA damage.

PubMed

Cui, Hongmei; Gu, Xinsheng; Chen, Jingshu; Xie, Ying; Ke, Sui; Wu, Jing; Golovko, Andrei; Morpurgo, Benjamin; Yan, Chunhong; Phillips, Timothy D; Xie, Wen; Luo, Jianyuan; Zhou, Zhijun; Tian, Yanan

2017-06-05

Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) significantly inhibited BaP-induced DNA damage and this protective effect was abolished in PXR-null hepatocytes. Mechanistically, PXR activation inhibited expression of AhR-target genes for CYP1A1, CYP1B1 and CYP1A2 that are required for BaP biotransformation in cultured liver cells, or in the livers of C57BL/6J mice. Using an AhR-responsive reporter assay as well as chromatin immunoprecipitation assay we found that PXR activation transcriptionally represses AhR-regulated gene expression. Furthermore, we found that PXR directly bound AhR at its DNA-binding domain, and this association may play a role in preventing of the AhR from binding to its target genes as shown in the ChIP assay. Taken together, our study has revealed a novel mechanism by which PXR protects liver cells from BaP-induced DNA damage through inhibiting the BaP biotransformation. Copyright © 2017 Elsevier B.V. All rights reserved.

Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome

PubMed Central

Kuwano, Yuki; Nishida, Kensei; Akaike, Yoko; Kurokawa, Ken; Nishikawa, Tatsuya; Masuda, Kiyoshi; Rokutan, Kazuhito

2016-01-01

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that phosphorylates and activates the apoptotic program through interaction with diverse downstream targets including tumor suppressor p53. HIPK2 is activated by genotoxic stimuli and modulates cell fate following DNA damage. The DNA damage response (DDR) is triggered by DNA lesions or chromatin alterations. The DDR regulates DNA repair, cell cycle checkpoint activation, and apoptosis to restore genome integrity and cellular homeostasis. Maintenance of the DDR is essential to prevent development of diseases caused by genomic instability, including cancer, defects of development, and neurodegenerative disorders. Recent studies reveal a novel HIPK2-mediated pathway for DDR through interaction with chromatin remodeling factor homeodomain protein 1γ. In this review, we will highlight the molecular mechanisms of HIPK2 and show its functions as a crucial DDR regulator. PMID:27689990

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

PubMed

Dutertre, Martin; Vagner, Stéphan

2017-10-27

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srinivas, L.; Shalini, V.K.

Twigs-dry leaves smoke condensate (TDS), as a source of clastogenic ROS and carcinogenic PAH, was investigated for its in vitro DNA-damaging effect in calf thymus DNA and human peripheral lymphocytes. An aqueous turmeric component--Aq.T--with an established antioxidant activity, was tested as a DNA protectant. TDS induced 13-fold damage to calf thymus DNA as judged by the emergence of a DNA damage specific, fluorescent product (em: 405 nm). Aq.T at 800 ng/microL extended 69% protection to calf thymus DNA and was comparable to the other protectants such as curcumin, BHA, vitamin E, SOD, and CAT. In human peripheral lymphocytes, TDS inducedmore » extensive DNA damage in comparison with the tumor promoter TPA, as judged by FADU. Aq.T at 300 ng/microL extended 90% protection to human lymphocyte DNA against TDS-induced damage, and was more effective than the other protectants--DABCO, D-mannitol, sodium benzoate, vitamin E (ROS quenchers), SOD, CAT (antioxidant enzymes), tannic acid, flufenamic acid, BHA, BHT, n-PG, curcumin and quercetin (antioxidants). Aq.T offered 65% protection to human lymphocyte DNA against TPA-induced damage and was comparable to SOD. The above results indicate that TDS induces substantial DNA damage in calf thymus DNA and human lymphocytes and Aq.T is an efficient protectant.« less

Lifestyle impacts on the aging associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

PubMed Central

Song, Zhangfa; von Figura, Guido; Liu, Yan; Kraus, Johann M.; Torrice, Chad; Dillon, Patric; Rudolph-Watabe, Masami; Ju, Zhenyu; Kestler, Hans A.; Sanoff, Hanna; Rudolph, K. Lenhard

2010-01-01

Summary Cellular aging is characterised by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and that activation of DNA damage responses. There is some evidence the DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging associated expression of serum markers of DNA damage (CRAMP, EF-1α, Stathmin, n-acetyl-glucosaminidase, and chitinase) in comparison to other described markers of cellular aging (p16INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age-independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age-independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16INK4a expression and negatively with telomere length in peripheral blood T-lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging. PMID:20560902

Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Junqiang; Doi, Hiroshi; Saar, Matthias

2013-12-01

Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome.more » The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.« less

Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways

PubMed Central

Silva, Bárbara Alcaraz; Stambaugh, Jessica R.

2013-01-01

Abstract. Telomeres are at the ends of chromosomes. Previous evidence suggests that laser-induced deoxyribose nucleic acid (DNA) breaks at chromosome ends during anaphase results in delayed cytokinesis. A possible explanation for this delay is that the DNA damage response (DDR) mechanism has been activated. We describe a live imaging method to study the effects of DDR activation following focal point near-infrared femtosecond laser microirradiation either at a single chromosome end or at a chromosome arm in mitotic anaphase cells. Laser microirradiation is used in combination with dual fluorescent labeling to monitor the co-localization of double-strand break marker γH2AX along with the DDR factors in PtK2 (Potorous tridactylus) cells. Laser-induced DNA breaks in chromosome ends as well as in chromosome arms results in recruitment of the following: poly(ADP-ribose) polymerase 1, checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80, and proliferating cell nuclear antigen. However, phosphorylated p53 at serine 15 is detected only at chromosome ends and not at chromosome arms. Full activation of DDR on damaged chromosome ends may explain previously published results that showed the delay of cytokinesis. PMID:24064949

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination.

PubMed

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S; Lan, Li

2015-07-07

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keeney, S.; Brody, T.; Linn, S.

1994-04-26

Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repairmore » defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo.« less

In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage

PubMed Central

Edwards, Sarah K.; Ono, Toshikazu; Wang, Shenliang; Jiang, Wei; Franzini, Raphael M.; Jung, Jong Wha; Chan, Ke Min; Kool, Eric T.

2015-01-01

The repair of oxidative damage to DNA is essential to avoidance of mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a chief source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report on the design and properties of fluorogenic probes that directly report on OGG1 (and bacterial homologue Fpg) activity in real time as the oxidized base is excised. The probes are short modified DNA oligomers containing fluorescent DNA bases and are designed to utilize the damaged DNA base itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probe designs containing these fluorophores, and we found an optimized probe OGR1 that yields a 60-fold light-up signal in vitro with OGG1 and Fpg, and can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes may be useful in quantifying enzyme activity and performing competitive inhibition assays. PMID:26073452

Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

PubMed

Singh, Deepti; Narayanamoorthy, Shwetha; Gamre, Sunita; Majumdar, Ananda Guha; Goswami, Manish; Gami, Umesh; Cherian, Susan; Subramanian, Mahesh

2018-05-20

Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles. Copyright © 2018 Elsevier Inc. All rights reserved.

Vitamin E Modifies High-Fat Diet-Induced Increase of DNA Strand Breaks, and Changes in Expression and DNA Methylation of Dnmt1 and MLH1 in C57BL/6J Male Mice.

PubMed

Remely, Marlene; Ferk, Franziska; Sterneder, Sonja; Setayesh, Tahereh; Kepcija, Tatjana; Roth, Sylvia; Noorizadeh, Rahil; Greunz, Martina; Rebhan, Irene; Wagner, Karl-Heinz; Knasmüller, Siegfried; Haslberger, Alexander

2017-06-14

Obesity is associated with low-grade inflammation, increased ROS production and DNA damage. Supplementation with antioxidants might ameliorate DNA damage and support epigenetic regulation of DNA repair. C57BL/6J male mice were fed a high-fat (HFD) or a control diet (CD) with and without vitamin E supplementation (4.5 mg/kg body weight (b.w.)) for four months. DNA damage, DNA promoter methylation and gene expression of Dnmt1 and a DNA repair gene ( MLH1 ) were assayed in liver and colon. The HFD resulted in organ specific changes in DNA damage, the epigenetically important Dnmt1 gene, and the DNA repair gene MLH1 . Vitamin E reduced DNA damage and showed organ-specific effects on MLH1 and Dnmt1 gene expression and methylation. These results suggest that interventions with antioxidants and epigenetic active food ingredients should be developed as an effective prevention for obesity-and oxidative stress-induced health risks.

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

PubMed Central

Cherubini, Gioia; Naim, Valeria; Caruso, Paola; Burla, Romina; Bogliolo, Massimo; Cundari, Enrico; Benihoud, Karim; Saggio, Isabella; Rosselli, Filippo

2011-01-01

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination. PMID:21421559

Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

PubMed

Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Manjeera, Dharani E; Dantuma, Nico P; Ström, Lena

2017-03-15

NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability. © 2017. Published by The Company of Biologists Ltd.

Effect of vitamin E (Tri E®) on antioxidant enzymes and DNA damage in rats following eight weeks exercise

PubMed Central

2011-01-01

Background Exercise is beneficial to health, but during exercise the body generates reactive oxygen species (ROS) which are known to result in oxidative stress. The present study analysed the effects of vitamin E (Tri E®) on antioxidant enzymes; superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (Cat) activity and DNA damage in rats undergoing eight weeks exercise. Methods Twenty four Sprague-Dawley rats (weighing 320-370 gm) were divided into four groups; a control group of sedentary rats which were given a normal diet, second group of sedentary rats with oral supplementation of 30 mg/kg/d of Tri E®, third group comprised of exercised rats on a normal diet, and the fourth group of exercised rats with oral supplementation of 30 mg/kg/d of Tri E®. The exercising rats were trained on a treadmill for 30 minutes per day for 8 weeks. Blood samples were taken before and after 8 weeks of the study to determine SOD, GPx, Cat activities and DNA damage. Results SOD activity decreased significantly in all the groups compared to baseline, however both exercised groups showed significant reduction in SOD activity as compared to the sedentary groups. Sedentary control groups showed significantly higher GPx and Cat activity compared to baseline and exercised groups. The supplemented groups, both exercised and non exercised groups, showed significant decrease in Cat activity as compared to their control groups with normal diet. DNA damage was significantly higher in exercising rats as compared to sedentary control. However in exercising groups, the DNA damage in supplemented group is significantly lower as compared to the non-supplemented group. Conclusions In conclusion, antioxidant enzymes activity were generally reduced in rats supplemented with Tri E® probably due to its synergistic anti-oxidative defence, as evidenced by the decrease in DNA damage in Tri E® supplemented exercise group. PMID:21513540

Effect of vitamin E (Tri E®) on antioxidant enzymes and DNA damage in rats following eight weeks exercise.

PubMed

Abd Hamid, Noor Aini; Hasrul, Mohd A; Ruzanna, Rusdiah J; Ibrahim, Ibrahim A; Baruah, Prasamit S; Mazlan, Musalmah; Yusof, Yasmin Anum Mohd; Ngah, Wan Zurinah Wan

2011-04-23

Exercise is beneficial to health, but during exercise the body generates reactive oxygen species (ROS) which are known to result in oxidative stress. The present study analysed the effects of vitamin E (Tri E®) on antioxidant enzymes; superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (Cat) activity and DNA damage in rats undergoing eight weeks exercise. Twenty four Sprague-Dawley rats (weighing 320-370 gm) were divided into four groups; a control group of sedentary rats which were given a normal diet, second group of sedentary rats with oral supplementation of 30 mg/kg/d of Tri E®, third group comprised of exercised rats on a normal diet, and the fourth group of exercised rats with oral supplementation of 30 mg/kg/d of Tri E®. The exercising rats were trained on a treadmill for 30 minutes per day for 8 weeks. Blood samples were taken before and after 8 weeks of the study to determine SOD, GPx, Cat activities and DNA damage. SOD activity decreased significantly in all the groups compared to baseline, however both exercised groups showed significant reduction in SOD activity as compared to the sedentary groups. Sedentary control groups showed significantly higher GPx and Cat activity compared to baseline and exercised groups. The supplemented groups, both exercised and non exercised groups, showed significant decrease in Cat activity as compared to their control groups with normal diet. DNA damage was significantly higher in exercising rats as compared to sedentary control. However in exercising groups, the DNA damage in supplemented group is significantly lower as compared to the non-supplemented group. In conclusion, antioxidant enzymes activity were generally reduced in rats supplemented with Tri E® probably due to its synergistic anti-oxidative defence, as evidenced by the decrease in DNA damage in Tri E® supplemented exercise group.

The role of DNA repair pathways in cisplatin resistant lung cancer.

PubMed

O'Grady, Shane; Finn, Stephen P; Cuffe, Sinead; Richard, Derek J; O'Byrne, Kenneth J; Barr, Martin P

2014-12-01

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA damage response in nephrotoxic and ischemic kidney injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Mingjuan; Tang, Chengyuan

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 reliesmore » on a thorough elucidation of the DDR pathways in various forms of AKI.« less

Trypanocidal nitroimidazole derivatives: relationships among chemical structure and genotoxic activity.

PubMed

Buschini, Annamaria; Giordani, Federica; de Albuquerque, Cristina Northfleet; Pellacani, Claudia; Pelosi, Giorgio; Rossi, Carlo; Zucchi, Tânia Maria Araújo Domingues; Poli, Paola

2007-05-15

Human American trypanosomiasis is resurgent in Latin Americans, and new drugs are urgently required as current medications suffer from a number of drawbacks. Some nitroheterocycles have been demonstrated to exert a potent activity against trypanosomes. However, host toxicity issues halted their development as trypanocides. As part of the efforts to develop new compounds in order to treat parasitic infections, it is important to define their structure-activity relationship. In this study, 5-nitromegazol and two of its analogues, 4-nitromegazol, and 1-methyl-5-nitro-2-imidazolecarboxaldehyde 5-nitroimidazole-thiosemicarbazone, were tested and compared for in vitro induction of DNA damage in human leukocytes by the comet assay, performed at different pHs to better identify the types of damage. Specific oxidatively generated damage to DNA was also measured by using the comet assay with endonucleases. DNA damage was found in 5-nitromegazol-treated cells: oxidative stress appeared as the main source of DNA damage. 4-Nitromegazol did not produce any significant effect, thus confirming that 4-nitroimidazoles isomers have no important biological activity. The 5-nitroimidazole-thiosemicarbazone induced DNA damage with a higher efficiency than 5-nitromegazol. The central role in the reduction process played by the acidic hydrazine proton present in the thiosemicarbazone group but not in the cyclic (thiadiazole) form can contribute to rationalise our results. Given its versatility, thiosemicarbazone moiety could be involved in different reactions with nitrogenous bases (nucleophilic and/or electrophilic attacks).

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

PubMed

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-11-02

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

PubMed Central

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-01-01

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway. PMID:26522181

Timing matters: error-prone gap filling and translesion synthesis in immunoglobulin gene hypermutation

PubMed Central

Sale, Julian E.; Batters, Christopher; Edmunds, Charlotte E.; Phillips, Lara G.; Simpson, Laura J.; Szüts, Dávid

2008-01-01

By temporarily deferring the repair of DNA lesions encountered during replication, the bypass of DNA damage is critical to the ability of cells to withstand genomic insults. Damage bypass can be achieved either by recombinational mechanisms that are generally accurate or by a process called translesion synthesis. Translesion synthesis involves replacing the stalled replicative polymerase with one of a number of specialized DNA polymerases whose active sites are able to tolerate a distorted or damaged DNA template. While this property allows the translesion polymerases to synthesize across damaged bases, it does so with the trade-off of an increased mutation rate. The deployment of these enzymes must therefore be carefully regulated. In addition to their important role in general DNA damage tolerance and mutagenesis, the translesion polymerases play a crucial role in converting the products of activation induced deaminase-catalysed cytidine deamination to mutations during immunoglobulin gene somatic hypermutation. In this paper, we specifically consider the control of translesion synthesis in the context of the timing of lesion bypass relative to replication fork progression and arrest at sites of DNA damage. We then examine how recent observations concerning the control of translesion synthesis might help refine our view of the mechanisms of immunoglobulin gene somatic hypermutation. PMID:19008194

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.

PubMed

Choi, Jun-Hyuk; Lindsey-Boltz, Laura A; Kemp, Michael; Mason, Aaron C; Wold, Marc S; Sancar, Aziz

2010-08-03

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

A Novel ATM/TP53/p21-Mediated Checkpoint Only Activated by Chronic γ-Irradiation

PubMed Central

Sasatani, Megumi; Iizuka, Daisuke; Masuda, Yuji; Inaba, Toshiya; Suzuki, Keiji; Ootsuyama, Akira; Umata, Toshiyuki; Kamiya, Kenji; Suzuki, Fumio

2014-01-01

Different levels or types of DNA damage activate distinct signaling pathways that elicit various cellular responses, including cell-cycle arrest, DNA repair, senescence, and apoptosis. Whereas a range of DNA-damage responses have been characterized, mechanisms underlying subsequent cell-fate decision remain elusive. Here we exposed cultured cells and mice to different doses and dose rates of γ-irradiation, which revealed cell-type-specific sensitivities to chronic, but not acute, γ-irradiation. Among tested cell types, human fibroblasts were associated with the highest levels of growth inhibition in response to chronic γ-irradiation. In this context, fibroblasts exhibited a reversible G1 cell-cycle arrest or an irreversible senescence-like growth arrest, depending on the irradiation dose rate or the rate of DNA damage. Remarkably, when the same dose of γ-irradiation was delivered chronically or acutely, chronic delivery induced considerably more cellular senescence. A similar effect was observed with primary cells isolated from irradiated mice. We demonstrate a critical role for the ataxia telangiectasia mutated (ATM)/tumor protein p53 (TP53)/p21 pathway in regulating DNA-damage-associated cell fate. Indeed, blocking the ATM/TP53/p21 pathway deregulated DNA damage responses, leading to micronucleus formation in chronically irradiated cells. Together these results provide insights into the mechanisms governing cell-fate determination in response to different rates of DNA damage. PMID:25093836

On binding specificity of (6-4) photolyase to a T(6-4)T DNA photoproduct*

NASA Astrophysics Data System (ADS)

Jepsen, Katrine Aalbæk; Solov'yov, Ilia A.

2017-06-01

Different factors lead to DNA damage and if it is not repaired in due time, the damaged DNA could initiate mutagenesis and cancer. To avoid this deadly scenario, specific enzymes can scavenge and repair the DNA, but the enzymes have to bind first to the damaged sites. We have investigated this binding for a specific enzyme called (6-4) photolyase, which is capable of repairing certain UV-induced damage in DNA. Through molecular dynamics simulations we describe the binding between photolyase and the DNA and reveal that several charged amino acid residues in the enzyme, such as arginines and lysines turn out to be important. Especially R421 is crucial, as it keeps the DNA strands at the damaged site inside the repair pocket of the enzyme separated. DNA photolyase is structurally highly homologous to a protein called cryptochrome. Both proteins are biologically activated similarly, namely through flavin co-factor photoexcitation. It is, however, striking that cryptochrome cannot repair UV-damaged DNA. The present investigation allowed us to conclude on the small but, apparently, critical differences between photolyase and cryptochrome. The performed analysis gives insight into important factors that govern the binding of UV-damaged DNA and reveal why cryptochrome cannot have this functionality.

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

PubMed Central

Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi

PubMed Central

Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo

2018-01-01

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control. PMID:29432450

Buckwheat Honey Attenuates Carbon Tetrachloride-Induced Liver and DNA Damage in Mice

PubMed Central

Cheng, Ni; Wu, Liming; Zheng, Jianbin; Cao, Wei

2015-01-01

Buckwheat honey, which is widely consumed in China, has a characteristic dark color. The objective of this study was to investigate the protective effects of buckwheat honey on liver and DNA damage induced by carbon tetrachloride in mice. The results revealed that buckwheat honey had high total phenolic content, and rutin, hesperetin, and p-coumaric acid were the main phenolic compounds present. Buckwheat honey possesses super DPPH radical scavenging activity and strong ferric reducing antioxidant power. Administration of buckwheat honey for 10 weeks significantly inhibited serum lipoprotein oxidation and increased serum oxygen radical absorbance capacity. Moreover, buckwheat honey significantly inhibited aspartate aminotransferase and alanine aminotransferase activities, which are enhanced by carbon tetrachloride. Hepatic malondialdehyde decreased and hepatic antioxidant enzymes (superoxide dismutase and glutathione peroxidase) increased in the presence of buckwheat honey. In a comet assay, lymphocyte DNA damage induced by carbon tetrachloride was significantly inhibited by buckwheat honey. Therefore, buckwheat honey has a hepatoprotective effect and inhibits DNA damage, activities that are primarily attributable to its high antioxidant capacity. PMID:26508989

Buckwheat Honey Attenuates Carbon Tetrachloride-Induced Liver and DNA Damage in Mice.

PubMed

Cheng, Ni; Wu, Liming; Zheng, Jianbin; Cao, Wei

2015-01-01

Buckwheat honey, which is widely consumed in China, has a characteristic dark color. The objective of this study was to investigate the protective effects of buckwheat honey on liver and DNA damage induced by carbon tetrachloride in mice. The results revealed that buckwheat honey had high total phenolic content, and rutin, hesperetin, and p-coumaric acid were the main phenolic compounds present. Buckwheat honey possesses super DPPH radical scavenging activity and strong ferric reducing antioxidant power. Administration of buckwheat honey for 10 weeks significantly inhibited serum lipoprotein oxidation and increased serum oxygen radical absorbance capacity. Moreover, buckwheat honey significantly inhibited aspartate aminotransferase and alanine aminotransferase activities, which are enhanced by carbon tetrachloride. Hepatic malondialdehyde decreased and hepatic antioxidant enzymes (superoxide dismutase and glutathione peroxidase) increased in the presence of buckwheat honey. In a comet assay, lymphocyte DNA damage induced by carbon tetrachloride was significantly inhibited by buckwheat honey. Therefore, buckwheat honey has a hepatoprotective effect and inhibits DNA damage, activities that are primarily attributable to its high antioxidant capacity.

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

The thyroid hormone receptor β induces DNA damage and premature senescence.

PubMed

Zambrano, Alberto; García-Carpizo, Verónica; Gallardo, María Esther; Villamuera, Raquel; Gómez-Ferrería, Maria Ana; Pascual, Angel; Buisine, Nicolas; Sachs, Laurent M; Garesse, Rafael; Aranda, Ana

2014-01-06

There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate-activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.

Genotoxic effects of environmental endocrine disruptors on the aquatic insect Chironomus riparius evaluated using the comet assay.

PubMed

Martínez-Paz, Pedro; Morales, Mónica; Martínez-Guitarte, José Luis; Morcillo, Gloria

2013-12-12

Genotoxicity is one of the most important toxic endpoints in chemical toxicity testing and environmental risk assessment. The aim of this study was to evaluate the genotoxic potential of various environmental pollutants frequently found in aquatic environments and characterized by their endocrine disrupting activity. Monitoring of DNA damage was undertaken after in vivo exposures of the aquatic larvae of the midge Chironomus riparius, a model organism that represents an abundant and ecologically relevant macroinvertebrate, widely used in freshwater toxicology. DNA-induced damage, resulting in DNA fragmentation, was quantified by the comet assay after short (24 h) and long (96 h) exposures to different concentrations of the selected toxicants: bisphenol A (BPA), nonylphenol (NP), pentachlorophenol (PCP), tributyltin (TBT) and triclosan (TCS). All five compounds were found to have genotoxic activity as demonstrated by significant increases in all the comet parameters (%DNA in tail, tail length, tail moment and Olive tail moment) at all tested concentrations. Persistent exposure did not increase the extent of DNA damage, except for TCS at the highest concentration, but generally there was a reduction in DNA damage thought to be associated with the induction of the detoxification processes and repairing mechanisms. Comparative analysis showed differences in the genotoxic potential between the chemicals, as well as significant time and concentration-dependent variations, which most likely reflect differences in the ability to repair DNA damage under the different treatments. The present report demonstrates the sensitivity of the benthic larvae of C. riparius to these environmental genotoxins suggesting its potential as biomonitor organism in freshwater ecosystems. The results obtained about the DNA-damaging potential of these environmental pollutants reinforce the need for additional studies on the genotoxicity of endocrine active substances that, by linking genotoxic activity to other biological responses, could provide further understanding of adverse effects in aquatic environments. Copyright © 2013 Elsevier B.V. All rights reserved.

A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis

PubMed Central

Li, Yanwen; Pelicic, Vladimir; Freemont, Paul S.; Baldwin, Geoff S.; Tang, Christoph M.

2013-01-01

Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair (BER) is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterised meningococcal BER enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence show that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes. PMID:22296581

Effects of physical exercise training in DNA damage and repair activity in humans with different genetic polymorphisms of hOGG1 (Ser326Cys).

PubMed

Soares, Jorge Pinto; Silva, Ana Inês; Silva, Amélia M; Almeida, Vanessa; Teixeira, João Paulo; Matos, Manuela; Gaivão, Isabel; Mota, Maria Paula

2015-12-01

The main purpose of this pilot study was to investigate the possible influence of genetic polymorphisms of the hOGG1 (Ser326Cys) gene in DNA damage and repair activity by 8-oxoguanine DNA glycosylase 1 (OGG1 enzyme) in response to 16 weeks of combined physical exercise training. Thirty-two healthy Caucasian men (40-74 years old) were enrolled in this study. All the subjects were submitted to a training of 16 weeks of combined physical exercise. The subjects with Ser/Ser genotype were considered as wild-type group (WTG), and Ser/Cys and Cys/Cys genotype were analysed together as mutant group (MG). We used comet assay in conjunction with formamidopyrimidine DNA glycoslyase (FPG) to analyse both strand breaks and FPG-sensitive sites. DNA repair activity were also analysed with the comet assay technique. Our results showed no differences between DNA damage (both strand breaks and FPG-sensitive sites) and repair activity (OGG1) between genotype groups (in the pre-training condition). Regarding the possible influence of genotype in the response to 16 weeks of physical exercise training, the results revealed a decrease in DNA strand breaks in both groups, a decrease in FPG-sensitive sites and an increase in total antioxidant capacity in the WTG, but no changes were found in MG. No significant changes in DNA repair activity was observed in both genotype groups with physical exercise training. This preliminary study suggests the possibility of different responses in DNA damage to the physical exercise training, considering the hOGG1 Ser326Cys polymorphism. Copyright © 2015 John Wiley & Sons, Ltd.

Integrated assessment of oxidative stress and DNA damage in earthworms (Eisenia fetida) exposed to azoxystrobin.

PubMed

Han, Yingnan; Zhu, Lusheng; Wang, Jinhua; Wang, Jun; Xie, Hui; Zhang, Shumin

2014-09-01

Azoxystrobin has been widely used in recent years. The present study investigated the oxidative stress and DNA damage effects of azoxystrobin on earthworms (Eisenia fetida). Earthworms were exposed to different azoxystrobin concentrations in an artificial soil (0, 0.1, 1, and 10mg/kg) and sampled on days 7, 14, 21, and 28. Superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione-S-transferase (GST), reactive oxygen species (ROS), and malondialdehyde (MDA) content were measured by an ultraviolet spectrophotometer to determine the antioxidant responses and lipid peroxidation. Single cell gel electrophoresis (SCGE) was used to detect DNA damage in the coelomocytes. Compared with these in the controls, earthworms exposed to azoxystrobin had excess ROS accumulation and greater SOD, POD, and GST activity while the opposite trend occurred for CAT activity. MDA content increased after 14-day exposure, and DNA damage was enhanced with an increase in the concentration of azoxystrobin. In conclusion, azoxystrobin caused oxidative stress leading to lipid peroxidation and DNA damage in earthworms. Copyright © 2014 Elsevier Inc. All rights reserved.

Activation of a yeast replication origin near a double-stranded DNA break.

PubMed

Raghuraman, M K; Brewer, B J; Fangman, W L

1994-03-01

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

DNA oxidative damage and life expectancy in houseflies.

PubMed Central

Agarwal, S; Sohal, R S

1994-01-01

The objective of this study was to explore the relationship between oxidative molecular damage and the aging process by determining whether such damage is associated with the rate of aging, using the adult housefly as the experimental organism. Because the somatic tissues in the housefly consist of long-lived postmitotic cells, it provides an excellent model system for studying cumulative age-related cellular alterations. Rate of aging in the housefly was manipulated by varying the rate of metabolism (physical activity). The concentration of 8-hydroxydeoxyguanosine (80HdG) was used as an indicator of DNA oxidation. Exposure of live flies to x-rays and hyperoxia elevated the level of 8OHdG. The level of 8OHdG in mitochondrial as well as total DNA increased with the age of flies. Mitochondrial DNA was 3 times more susceptible to age-related oxidative damage than nuclear DNA. A decrease in the level of physical activity of the flies was found to prolong the life-span and corresponding reduce the level of 8OHdG in both mitochondrial and total DNA. Under all conditions examined, mitochondrial DNA exhibited a higher level of oxidative damage than total DNA. The 8OHdG levels were found to be inversely associated with the life expectancy of houseflies. The pattern of age-associated accrural of 8OHdG was virtually identical to that of protein carbonyl content. Altoghether, results of this study support the hypothesis that oxidative molecular damage is a causal factor in senescence. PMID:7991627

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

PubMed

Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C M; Jansen, Jacob G; Hogenbirk, Marc A; de Wind, Niels; Jacobs, Heinz

2015-01-01

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

PubMed Central

Szyjka, Shawn J.; Aparicio, Jennifer G.; Viggiani, Christopher J.; Knott, Simon; Xu, Weihong; Tavaré, Simon; Aparicio, Oscar M.

2008-01-01

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2–Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Δ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair. PMID:18628397

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Hippo pathway and protection of genome stability in response to DNA damage.

PubMed

Pefani, Dafni E; O'Neill, Eric

2016-04-01

The integrity of DNA is constantly challenged by exposure to the damaging effects of chemical and physical agents. Elucidating the cellular mechanisms that maintain genomic integrity via DNA repair and cell growth control is vital because errors in these processes lead to genomic damage and the development of cancer. By gaining a deep molecular understanding of the signaling pathways regulating genome integrity it is hoped to uncover new therapeutics and treatment designs to combat cancer. Components of the Hippo pathway, a tumor-suppressor cascade, have recently been defined to limit cancer transformation in response to DNA damage. In this review, we briefly introduce the Hippo signaling cascade in mammals and discuss in detail how the Hippo pathway has been established as part of the DNA damage response, activated by apical signaling kinases that recognize breaks in DNA. We also highlight the significance of the Hippo pathway activator RASSF1A tumor suppressor, a direct target of ataxia telangiectasia mutated and ataxia telangiectasia and Rad3 related ATR. Furthermore we discuss how Hippo pathway in response DNA lesions can induce cell death via Yes-associated protein (YAP) (the canonical Hippo pathway effector) or promote maintenance of genome integrity in a YAP-independent manner. © 2015 FEBS.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

PubMed Central

Park, Sojin; Choi, Seoyun; Ahn, Byungchan

2016-01-01

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

PubMed

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Mangiferin activates the Nrf2-ARE pathway and reduces etoposide-induced DNA damage in human umbilical cord mononuclear blood cells.

PubMed

Zhang, Benping; Zhao, Jie; Li, Shanshan; Zeng, Linglan; Chen, Yan; Fang, Jun

2015-04-01

Mangiferin (2-C-β-d-gluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) is a well-known natural antioxidant distributed in various plants of the Anacardiaceae and Gentianaceae families. Mangiferin can inhibit carcinogen-induced lung or colon tumor formation in experimental animals. However, the molecular mechanisms of its chemopreventive activity remain unexplored. This study aimed to investigate the effects of mangiferin on chemical carcinogen-induced DNA damage and Nrf2-ARE signaling in hematopoietic cells. Mononuclear cells (MNCs) were isolated from human umbilical cord blood (hUCB). DNA damage was evaluated by comet and micronucleus assays. The expression of Nrf2 and NQO1 was examined by immunofluorescence and western blotting. An electrophoretic mobility shift assay (EMSA) was used to detect the binding activity of Nrf2 with NQO1-ARE sequences. We found that mangiferin treatment significantly reduced DNA damage in etoposide-treated MNCs, which was verified by decreased olive tail moment (OTM) and micronucleus (MN) frequency. Mangiferin treatment significantly promoted Nrf2 translocation into the nucleus and increased nuclear Nrf2 expression. Moreover, NQO1, an Nrf2 signaling target, was significantly upregulated by mangiferin treatment, and the binding activity of Nrf2 with NQO1-ARE sequences was elevated after mangiferin treatment. Mangiferin activated Nrf2 signaling, upregulated NQO1 expression, and significantly reduced etoposide-induced DNA damage. Thus, mangiferin is a potential cytoprotective agent for hematopoietic cells.

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

PubMed

Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

2015-07-01

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

House dust mite-induced asthma causes oxidative damage and DNA double-strand breaks in the lungs.

PubMed

Chan, Tze Khee; Loh, Xin Yi; Peh, Hong Yong; Tan, W N Felicia; Tan, W S Daniel; Li, Na; Tay, Ian J J; Wong, W S Fred; Engelward, Bevin P

2016-07-01

Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) in vivo and in vitro. We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX) foci in bronchial epithelium. BEAS-2B cells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart.

PubMed

Gredilla, R; Barja, G; López-Torres, M

2001-10-01

Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T4) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks. Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA. These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.

Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells.

PubMed

You, B-J; Wu, Y-C; Lee, C-L; Lee, H-Z

2014-03-01

4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

6-Gingerol prevents MEHP-induced DNA damage in human umbilical vein endothelia cells.

PubMed

Yang, G; Gao, X; Jiang, L; Sun, X; Liu, X; Chen, M; Yao, X; Sun, Q; Wang, S

2017-11-01

Mono (2-ethylhexyl) phthalate (MEHP) is the principal metabolite of di (2-etylhexyl) phthalate, which is widely used as a plasticizer, especially in medical devices. MEHP has toxic effects on cardiovascular system. The aim of this study was to investigate the possibility that 6-gingerol may inhibit the oxidative DNA damage of MEHP in human umbilical vein endothelial cells (HUVECs) and the potential mechanism. The comet assay was used to monitor DNA strand breaks. We have shown that 6-gingerol significantly reduced the DNA strand breaks caused by MEHP. MEHP increased the levels of reactive oxygen species and malondialdehyde, decreased the level of glutathione and activity of superoxide dismutase, and altered the mitochondrial membrane potential. In addition, DNA damage-associated proteins (p53 and p-Chk2 (T68)) were significantly increased by the treatment of MEHP. Those effects can all be protected by 6-gingerol. The results firmly indicate that 6-gingerol may have a strong protective ability against the DNA damage caused by MEHP in HUVECs, and the mechanism may relate to the antioxidant activity.

Approaches for characterizing threshold dose-response relationships for DNA-damage pathways involved in carcinogenicity in vivo and micronuclei formation in vitro.

PubMed

Clewell, Rebecca A; Andersen, Melvin E

2016-05-01

Assessing the shape of dose-response curves for DNA-damage in cellular systems and for the consequences of DNA damage in intact animals remains a controversial topic. This overview looks at aspects of the pharmacokinetics (PK) and pharmacodynamics (PD) of cellular DNA-damage/repair and their role in defining the shape of dose-response curves using an in vivo example with formaldehyde and in vitro examples for micronuclei (MN) formation with several test compounds. Formaldehyde is both strongly mutagenic and an endogenous metabolite in cells. With increasing inhaled concentrations, there were transitions in gene changes, from activation of selective stress pathway genes at low concentrations, to activation of pathways for cell-cycle control, p53-DNA damage, and stem cell niche pathways at higher exposures. These gene expression changes were more consistent with dose-dependent transitions in the PD responses to formaldehyde in epithelial cells in the intact rat rather than the low-dose linear extrapolation methods currently used for carcinogens. However, more complete PD explanations of non-linear dose response for creation of fixed damage in cells require detailed examination of cellular responses in vitro using measures of DNA damage and repair that are not easily accessible in the intact animal. In the second section of the article, we illustrate an approach from our laboratory that develops fit-for-purpose, in vitro assays and evaluates the PD of DNA damage and repair through studies using prototypical DNA-damaging agents. Examination of a broad range of responses in these cells showed that transcriptional upregulation of cell cycle control and DNA repair pathways only occurred at doses higher than those causing overt damage fixed damage-measured as MN formation. Lower levels of damage appear to be handled by post-translational repair process using pre-existing proteins. In depth evaluation of the PD properties of one such post-translational process (formation of DNA repair centers; DRCs) has indicated that the formation of DRCs and their ability to complete repair before replication are consistent with threshold behaviours for mutagenesis and, by extension, with chemical carcinogenesis. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Quinone Based Antitumor Agent Sepantronium Bromide (YM155) Causes Oxygen Independent Redox Activated Oxidative DNA Damage.

PubMed

Wani, Tasaduq Hussain; Surendran, Sreeraj; Jana, Anal; Chakrabarty, Anindita; Chowdhury, Goutam

2018-06-13

Sepantronium bromide (YM155) is a small molecule antitumor agent currently in phase II clinical trials. Although developed as survivin suppressor, YM155's primary mode of action has recently been found to be DNA damage. However, the mechanism of DNA damage by YM155 is still unknown. Knowing the mechanism of action of an anticancer drug is necessary to formulate a rational drug combination and select a cancer type for achieving maximum clinical efficacy. Using cell-based assays we showed that YM155 cause extensive DNA cleavage and reactive oxygen species generation. DNA cleavage by YM155 was found to be inhibited by radical scavengers and desferal. The reducing agent DTT and the cellular reducing system xanthine/xanthine oxidase were found to reductively activate YM155 and cause DNA cleavage. Unlike quinones, DNA cleavage by YM155 occurs in the presence of catalase and under hypoxic conditions indicating that hydrogen peroxide and oxygen is not necessary. Although YM155 is a quinone, it does not follow a typical quinone mechanism. Consistent with these observations a mechanism has been proposed that suggests that YM155 can cause oxidative DNA cleavage upon two electron reductive activation.

Chitinase enzyme activity in CSF is a powerful biomarker of Alzheimer disease.

PubMed

Watabe-Rudolph, M; Song, Z; Lausser, L; Schnack, C; Begus-Nahrmann, Y; Scheithauer, M-O; Rettinger, G; Otto, M; Tumani, H; Thal, D R; Attems, J; Jellinger, K A; Kestler, H A; von Arnim, C A F; Rudolph, K L

2012-02-21

DNA damage accumulation in brain is associated with the development of Alzheimer disease (AD), but newly identified protein markers of DNA damage have not been evaluated in the diagnosis of AD and other forms of dementia. Here, we analyzed the level of novel biomarkers of DNA damage and telomere dysfunction (chitinase activity, N-acetyl-glucosaminidase activity, stathmin, and EF-1α) in CSF of 94 patients with AD, 41 patients with non-AD dementia, and 40 control patients without dementia. Enzymatic activity of chitinase (chitotriosidase activity) and stathmin protein level were significantly increased in CSF of patients with AD and non-AD dementia compared with that of no dementia control patients. As a single marker, chitinase activity was most powerful for distinguishing patients with AD from no dementia patients with an accuracy of 85.8% using a single threshold. Discrimination was even superior to clinically standard CSF markers that showed an accuracy of 78.4% (β-amyloid) and 77.6% (tau). Combined analysis of chitinase with other markers increased the accuracy to a maximum of 91%. The biomarkers of DNA damage were also increased in CSF of patients with non-AD dementia compared with no dementia patients, and the new biomarkers improved the diagnosis of non-AD dementia as well as the discrimination of AD from non-AD dementia. Taken together, the findings in this study provide experimental evidence that DNA damage markers are significantly increased in AD and non-AD dementia. The biomarkers identified outperformed the standard CSF markers for diagnosing AD and non-AD dementia in the cohort investigated.

Activation of DNA damage repair pathways by murine polyomavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiser, Katie; Nicholas, Catherine; Garcea, Robert

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling.more » ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.« less

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage

PubMed Central

Svilar, David; Goellner, Eva M.; Almeida, Karen H.

2011-01-01

Abstract Nuclear and mitochondrial genomes are under continuous assault by a combination of environmentally and endogenously derived reactive oxygen species, inducing the formation and accumulation of mutagenic, toxic, and/or genome-destabilizing DNA lesions. Failure to resolve these lesions through one or more DNA-repair processes is associated with genome instability, mitochondrial dysfunction, neurodegeneration, inflammation, aging, and cancer, emphasizing the importance of characterizing the pathways and proteins involved in the repair of oxidative DNA damage. This review focuses on the repair of oxidative damage–induced lesions in nuclear and mitochondrial DNA mediated by the base excision repair (BER) pathway in mammalian cells. We discuss the multiple BER subpathways that are initiated by one of 11 different DNA glycosylases of three subtypes: (a) bifunctional with an associated β-lyase activity; (b) monofunctional; and (c) bifunctional with an associated β,δ-lyase activity. These three subtypes of DNA glycosylases all initiate BER but yield different chemical intermediates and hence different BER complexes to complete repair. Additionally, we briefly summarize alternate repair events mediated by BER proteins and the role of BER in the repair of mitochondrial DNA damage induced by ROS. Finally, we discuss the relation of BER and oxidative DNA damage in the onset of human disease. Antioxid. Redox Signal. 14, 2491–2507. PMID:20649466

Oxidative stress and genetic damage among workers exposed primarily to organophosphate and pyrethroid pesticides.

PubMed

Zepeda-Arce, Rigoberto; Rojas-García, Aurora Elizabeth; Benitez-Trinidad, Alma; Herrera-Moreno, José Francisco; Medina-Díaz, Irma Martha; Barrón-Vivanco, Briscia S; Villegas, Germán Pier; Hernández-Ochoa, Isabel; Sólis Heredia, María de Jesús; Bernal-Hernández, Yael Y

2017-06-01

The indiscriminate use of pesticides in agriculture and public health campaigns has been associated with an increase of oxidative stress and DNA damage, resulting in health outcomes. Some defense mechanisms against free radical-induced oxidative damage include the antioxidant enzyme systems. The aim of this study was to determine the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and the relationship of antioxidant enzyme levels with DNA damage among sprayers (workers) occupationally exposed to pesticides. The determinations of MDA and antioxidant enzymes were performed spectrophotometrically. The genotoxic effects were evaluated using the comet assay. The results showed a marginally significant decrease in SOD and CAT activities in the high exposure group compared to the control group. For MDA, statistically significant differences were found among people working long term vs. those working temporarily (P = 0.02) as sprayers. In the moderate exposure group, a positive correlation was observed between MDA levels and GPx activity. In the high exposure group, a negative correlation was observed between GR and CAT activities, and between MDA levels and GPx activities. Furthermore, in the high exposure group, a positive correlation between DNA damage parameters and MDA levels was observed. The results suggest an important role of antioxidant enzymes for the protection of DNA damage caused by occupational exposure to pesticides. © 2017 Wiley Periodicals, Inc.

Involvement of DNA polymerase beta in repairing oxidative damages induced by antitumor drug adriamycin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Shukun; Wu Mei; Zhang Zunzhen, E-mail: zhangzunzhen@163.co

2010-08-01

Adriamycin (ADM) is a widely used antineoplastic drug. However, the increasing cellular resistance has become a serious limitation to ADM clinical application. The most important mechanism related to ADM-induced cell death is oxidative DNA damage mediated by reactive oxygen species (ROS). Base excision repair (BER) is a major pathway in the repair of DNA single strand break (SSB) and oxidized base. In this study, we firstly applied the murine embryo fibroblasts wild-type (pol {beta} +/+) and homozygous pol {beta} null cell (pol {beta} -/-) as a model to investigate ADM DNA-damaging effects and the molecular basis underlying these effects. Here,more » cellular sensitivity to ADM was examined using colorimetric assay and colony forming assay. ADM-induced cellular ROS level and the alteration of superoxide dismutase (SOD) activity were measured by commercial kits. Further, DNA strand break, chromosomal damage and gene mutation were assessed by comet assay, micronucleus test and hprt gene mutation assay, respectively. The results showed that pol {beta} -/- cells were more sensitive to ADM compared with pol {beta} +/+ cells and more severe SSB and chromosomal damage as well as higher hprt gene mutation frequency were observed in pol {beta} -/- cells. ROS level in pol {beta} -/- cells increased along with decreased activity of SOD. These results demonstrated that pol {beta} deficiency could enable ROS accumulation with SOD activity decrease, further elevate oxidative DNA damage, and subsequently result in SSB, chromosome cleavage as well as gene mutation, which may be partly responsible for the cytotoxicity of ADM and the hypersensitivity of pol {beta} -/- cells to ADM. These findings suggested that pol {beta} is vital for repairing oxidative damage induced by ADM.« less

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

Mitochondrial Cardiomyopathy Caused by Elevated Reactive Oxygen Species and Impaired Cardiomyocyte Proliferation.

PubMed

Zhang, Donghui; Li, Yifei; Heims-Waldron, Danielle; Bezzerides, Vassilios; Guatimosim, Silvia; Guo, Yuxuan; Gu, Fei; Zhou, Pingzhu; Lin, Zhiqiang; Ma, Qing; Liu, Jianming; Wang, Da-Zhi; Pu, William T

2018-01-05

Although mitochondrial diseases often cause abnormal myocardial development, the mechanisms by which mitochondria influence heart growth and function are poorly understood. To investigate these disease mechanisms, we studied a genetic model of mitochondrial dysfunction caused by inactivation of Tfam (transcription factor A, mitochondrial), a nuclear-encoded gene that is essential for mitochondrial gene transcription and mitochondrial DNA replication. Tfam inactivation by Nkx2.5 Cre caused mitochondrial dysfunction and embryonic lethal myocardial hypoplasia. Tfam inactivation was accompanied by elevated production of reactive oxygen species (ROS) and reduced cardiomyocyte proliferation. Mosaic embryonic Tfam inactivation confirmed that the block to cardiomyocyte proliferation was cell autonomous. Transcriptional profiling by RNA-seq demonstrated the activation of the DNA damage pathway. Pharmacological inhibition of ROS or the DNA damage response pathway restored cardiomyocyte proliferation in cultured fetal cardiomyocytes. Neonatal Tfam inactivation by AAV9-cTnT-Cre caused progressive, lethal dilated cardiomyopathy. Remarkably, postnatal Tfam inactivation and disruption of mitochondrial function did not impair cardiomyocyte maturation. Rather, it elevated ROS production, activated the DNA damage response pathway, and decreased cardiomyocyte proliferation. We identified a transient window during the first postnatal week when inhibition of ROS or the DNA damage response pathway ameliorated the detrimental effect of Tfam inactivation. Mitochondrial dysfunction caused by Tfam inactivation induced ROS production, activated the DNA damage response, and caused cardiomyocyte cell cycle arrest, ultimately resulting in lethal cardiomyopathy. Normal mitochondrial function was not required for cardiomyocyte maturation. Pharmacological inhibition of ROS or DNA damage response pathways is a potential strategy to prevent cardiac dysfunction caused by some forms of mitochondrial dysfunction. © 2017 American Heart Association, Inc.

A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage.

PubMed

Kubiak, Jeffrey M; Culyba, Matthew J; Liu, Monica Yun; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2017-11-17

The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes autoproteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway.

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

PubMed

Mukherjee, Anirban; Vasquez, Karen M

2011-08-01

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Preterm newborns show slower repair of oxidative damage and paternal smoking associated DNA damage.

PubMed

Vande Loock, Kim; Ciardelli, Roberta; Decordier, Ilse; Plas, Gina; Haumont, Dominique; Kirsch-Volders, Micheline

2012-09-01

Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero, increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H(2)O(2) induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity.

CDK1 promotes nascent DNA synthesis and induces resistance of cancer cells to DNA-damaging therapeutic agents

PubMed Central

Liao, Hongwei; Ji, Fang; Geng, Xinwei; Xing, Meichun; Li, Wen; Chen, Zhihua; Shen, Huahao; Ying, Songmin

2017-01-01

Cyclin dependent kinase 1 (CDK1) is essential for cell viability and plays a vital role in many biological events including cell cycle control, DNA damage repair, and checkpoint activation. Here, we identify an unanticipated role for CDK1 in promoting nascent DNA synthesis during S-phase. We report that a short duration of CDK1 inhibition, which does not perturb cell cycle progression, triggers a replication-associated DNA damage response (DDR). This DDR is associated with a disruption of replication fork progression and leads to genome instability. Moreover, we show that compromised CDK1 activity dramatically increases the efficacy of chemotherapeutic agents that kill cancer cells through perturbing DNA replication, including Olaparib, an FDA approved PARP inhibitor. Our study has revealed an important role for CDK1 in the DNA replication program, and suggests that the therapeutic targeting CDK1 may be a novel approach for combination chemotherapy. PMID:29207595

Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

PubMed Central

Cruz-Gregorio, Alfredo; Manzo-Merino, Joaquín; Gonzaléz-García, María Cecilia; Pedraza-Chaverri, José; Medina-Campos, Omar Noel; Valverde, Mahara; Rojas, Emilio; Rodríguez-Sastre, María Alexandra; García-Cuellar, Claudia María; Lizano, Marcela

2018-01-01

Oxidative stress has been proposed as a risk factor for cervical cancer development. However, few studies have evaluated the redox state associated with human papillomavirus (HPV) infection. The aim of this work was to determine the role of the early expressed viral proteins E1, E2, E6 and E7 from HPV types 16 and 18 in the modulation of the redox state in an integral form. Therefore, generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH), levels and activity of the antioxidant enzymes catalase and superoxide dismutase (SOD) and deoxyribonucleic acid (DNA) damage, were analysed in epithelial cells ectopically expressing the viral proteins. Our research shows that E6 oncoproteins decreased GSH and catalase protein levels, as well as its enzymatic activity, which was associated with an increase in ROS production and DNA damage. In contrast, E7 oncoproteins increased GSH, as well as catalase protein levels and its activity, which correlated with a decrease in ROS without affecting DNA integrity. The co-expression of both E6 and E7 oncoproteins neutralized the effects that were independently observed for each of the viral proteins. Additionally, the combined expression of E1 and E2 proteins increased ROS levels with the subsequent increase in the marker for DNA damage phospho-histone 2AX (γH2AX). A decrease in GSH, as well as SOD2 levels and activity were also detected in the presence of E1 and E2, even though catalase activity increased. This study demonstrates that HPV early expressed proteins differentially modulate cellular redox state and DNA damage. PMID:29483822

The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA

PubMed Central

Gewandter, Jennifer S; Bambara, Robert A

2011-01-01

DNA damage, stalled replication forks, errors in mRNA splicing and availability of nutrients activate specific phosphatidylinositiol-3-kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2 or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA. PMID:21701263

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

Femtosecond near-infrared laser microirradiation reveals a crucial role for PARP signaling on factor assemblies at DNA damage sites

PubMed Central

Saquilabon Cruz, Gladys Mae; Kong, Xiangduo; Silva, Bárbara Alcaraz; Khatibzadeh, Nima; Thai, Ryan; Berns, Michael W.; Yokomori, Kyoko

2016-01-01

Laser microirradiation is a powerful tool for real-time single-cell analysis of the DNA damage response (DDR). It is often found, however, that factor recruitment or modification profiles vary depending on the laser system employed. This is likely due to an incomplete understanding of how laser conditions/dosages affect the amounts and types of damage and the DDR. We compared different irradiation conditions using a femtosecond near-infrared laser and found distinct damage site recruitment thresholds for 53BP1 and TRF2 correlating with the dose-dependent increase of strand breaks and damage complexity. Low input-power microirradiation that induces relatively simple strand breaks led to robust recruitment of 53BP1 but not TRF2. In contrast, increased strand breaks with complex damage including crosslinking and base damage generated by high input-power microirradiation resulted in TRF2 recruitment to damage sites with no 53BP1 clustering. We found that poly(ADP-ribose) polymerase (PARP) activation distinguishes between the two damage states and that PARP activation is essential for rapid TRF2 recruitment while suppressing 53BP1 accumulation at damage sites. Thus, our results reveal that careful titration of laser irradiation conditions allows induction of varying amounts and complexities of DNA damage that are gauged by differential PARP activation regulating protein assembly at the damage site. PMID:26424850

Choline deficiency increases lymphocyte apoptosis and DNA damage in humans.

PubMed

da Costa, Kerry-Ann; Niculescu, Mihai D; Craciunescu, Corneliu N; Fischer, Leslie M; Zeisel, Steven H

2006-07-01

Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans. The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans. Fifty-one men and women aged 18-70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138-550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase-mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays. All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet. A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dysfunction also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected.

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

PubMed Central

Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

2016-01-01

Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

PubMed Central

Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

2013-01-01

Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. PMID:23523584

Cidofovir is active against human papillomavirus positive and negative head and neck and cervical tumor cells by causing DNA damage as one of its working mechanisms.

PubMed

Mertens, Barbara; Nogueira, Tatiane; Stranska, Ruzena; Naesens, Lieve; Andrei, Graciela; Snoeck, Robert

2016-07-26

Human papillomavirus (HPV) causes cervical cancer and a large fraction of head and neck squamous cell carcinomas (HNSCC). Cidofovir (CDV) proved efficacious in the treatment of several HPV-induced benign and malignant hyper proliferations. To provide a better insight into how CDV selectively eradicates transformed cells, HPV+ and HPV- cervical carcinoma and HNSCC cell lines were compared to normal cells for antiproliferative effects, CDV metabolism, drug incorporation into cellular DNA, and DNA damage. Incorporation of CDV into cellular DNA was higher in tumor cells than in normal cells and correlated with CDV antiproliferative effects, which were independent of HPV status. Increase in phospho-ATM levels was detected following CDV exposure and higher levels of γ-H2AX (a quantitative marker of double-strand breaks) were measured in tumor cells compared to normal cells. A correlation between DNA damage and CDV incorporation into DNA was found but not between DNA damage and CDV antiproliferative effects. These data indicate that CDV antiproliferative effects result from incorporation of the drug into DNA causing DNA damage. However, the anti-tumor effects of CDV cannot be exclusively ascribed to DNA damage. Furthermore, CDV can be considered a promising broad spectrum anti-cancer agent, not restricted to HPV+ lesions.

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

PubMed Central

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A.; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S.; Lan, Li

2015-01-01

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome. PMID:26100862

Autophagy and genomic integrity

PubMed Central

Vessoni, A T; Filippi-Chiela, E C; Menck, C FM; Lenz, G

2013-01-01

DNA lesions, constantly produced by endogenous and exogenous sources, activate the DNA damage response (DDR), which involves detection, signaling and repair of the damage. Autophagy, a lysosome-dependent degradation pathway that is activated by stressful situations such as starvation and oxidative stress, regulates cell fate after DNA damage and also has a pivotal role in the maintenance of nuclear and mitochondrial genomic integrity. Here, we review important evidence regarding the role played by autophagy in preventing genomic instability and tumorigenesis, as well as in micronuclei degradation. Several pathways governing autophagy activation after DNA injury and the influence of autophagy upon the processing of genomic lesions are also discussed herein. In this line, the mechanisms by which several proteins participate in both DDR and autophagy, and the importance of this crosstalk in cancer and neurodegeneration will be presented in an integrated fashion. At last, we present a hypothetical model of the role played by autophagy in dictating cell fate after genotoxic stress. PMID:23933813

Attenuation of Cisplatin-Induced Neurotoxicity by Cyanidin, a Natural Inhibitor of ROS-Mediated Apoptosis in PC12 Cells.

PubMed

Li, Da-wei; Sun, Jing-yi; Wang, Kun; Zhang, Shuai; Hou, Ya-jun; Yang, Ming-feng; Fu, Xiao-yan; Zhang, Zong-yong; Mao, Lei-lei; Yuan, Hui; Fang, Jie; Fan, Cun-dong; Zhu, Mei-jia; Sun, Bao-liang

2015-10-01

Cisplatin-based chemotherapy in clinic is severely limited by its adverse effect, including neurotoxicity. Oxidative damage contributes to cisplatin-induced neurotoxicity, but the mechanism remains unclearly. Cyanidin, a natural flavonoid compound, exhibits powerful antioxidant activity. Hence, we investigated the protective effects of cyanidin on PC12 cells against cisplatin-induced neurotoxicity and explored the underlying mechanisms. The results showed that cisplatin-induced cytotoxicity was completely reversed by cyanidin through inhibition of PC12 cell apoptosis, as proved by the attenuation of Sub-G1 peak, PARP cleavage, and caspases-3 activation. Mechanistically, cyanidin significantly inhibited reactive oxygen species (ROS)-induced DNA damage in cisplatin-treated PC12 cells. Our findings revealed that cyanidin as an apoptotic inhibitor effectively blocked cisplatin-induced neurotoxicity through inhibition of ROS-mediated DNA damage and apoptosis, predicating its therapeutic potential in prevention of chemotherapy-induced neurotoxicity. Cisplatin caused DNA damage, activated p53, and subsequently induced PC12 cells apoptosis by triggering ROS overproduction. However, cyanidin administration effectively inhibited DNA damage, attenuated p53 phosphorylation, and eventually reversed cisplatin-induced PC12 cell apoptosis through inhibition ROS accumulation.

Genoprotective and hepatoprotective effects of Guarana (Paullinia cupana Mart. var. sorbilis) on CCl4-induced liver damage in rats.

PubMed

Kober, Helena; Tatsch, Etiane; Torbitz, Vanessa Dorneles; Cargnin, Lara Peruzzolo; Sangoi, Manuela Borges; Bochi, Guilherme Vargas; da Silva, Andreia Regina Haas; Barbisan, Fernanda; Ribeiro, Euler Esteves; da Cruz, Ivana Beatrice Mânica; Moresco, Rafael Noal

2016-01-01

Several biological effects of Paullinia cupana (guarana) have been demonstrated, but little information is available on its effects on the liver. The current study was designed to evaluate the hepatoprotective and genoprotective effects of powder seeds from guarana on CCl4-induced liver injury in rats. Male Wistar rats were pretreated with guarana powder (100, 300 and 600 mg/kg) or silymarin 100 mg/kg daily for 14 days before treatment with a single dose of CCl4 (50% CCl4, 1 mL/kg, intraperitoneally). The treatment with CCl4 significantly increased the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In addition, CCl4 increased the DNA damage index in hepatocytes. Guarana in all concentrations was effective in decreasing the ALT and AST activities when compared with the CCl4-treated group. The treatment with guarana decreased DNA damage index when compared with the CCl4-treated group. In addition, the DNA damage index showed a significant positive correlation with AST and ALT. These results indicate that the guarana has hepatoprotective activity and prevents the DNA strand breakage in the CCl4-induced liver damage in rats.

Unexpected Function of the Glucanosyltransferase Gas1 in the DNA Damage Response Linked to Histone H3 Acetyltransferases in Saccharomyces cerevisiae

PubMed Central

Eustice, Moriah; Pillus, Lorraine

2014-01-01

Chromatin organization and structure are crucial for transcriptional regulation, DNA replication, and damage repair. Although initially characterized in remodeling cell wall glucans, the β-1,3-glucanosyltransferase Gas1 was recently discovered to regulate transcriptional silencing in a manner separable from its activity at the cell wall. However, the function of Gas1 in modulating chromatin remains largely unexplored. Our genetic characterization revealed that GAS1 had critical interactions with genes encoding the histone H3 lysine acetyltransferases Gcn5 and Sas3. Specifically, whereas the gas1gcn5 double mutant was synthetically lethal, deletion of both GAS1 and SAS3 restored silencing in Saccharomyces cerevisiae. The loss of GAS1 also led to broad DNA damage sensitivity with reduced Rad53 phosphorylation and defective cell cycle checkpoint activation following exposure to select genotoxins. Deletion of SAS3 in the gas1 background restored both Rad53 phosphorylation and checkpoint activation following exposure to genotoxins that trigger the DNA replication checkpoint. Our analysis thus uncovers previously unsuspected functions for both Gas1 and Sas3 in DNA damage response and cell cycle regulation. PMID:24532730

NOTCH1 Inhibits Activation of ATM by Impairing the Formation of an ATM-FOXO3a-KAT5/Tip60 Complex.

PubMed

Adamowicz, Marek; Vermezovic, Jelena; d'Adda di Fagagna, Fabrizio

2016-08-23

The DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Rapid communications: antiperspirant induced DNA damage in canine cells by comet assay.

PubMed

Yiu, Gloria

2004-01-01

Abstract Millions of people around the world use antiperspirants to decrease or eliminate body odors. Most antiperspirants contain aluminum zirconium or another form of aluminum as its active ingredient. The present investigation applied Comet assay to detect if Secret Platinum for women, Old Spice for men, or Crystal Natural produced DNA damage in Madin-Darby canine kidney cells (MDCKII). This study has shown that antiperspirants cause DNA damage on a single-cell level. Additionally, our data showed us that in general, Secret Platinum for women and Old Spice for men, produced equivalent damage. Crystal Natural, marketed as being safer or less damaging, induced the most extensive damage of all three antiperspirants tested.

Activation of EGFR and ERBB2 by Helicobacter pylori results in survival of gastric epithelial cells with DNA damage.

PubMed

Chaturvedi, Rupesh; Asim, Mohammad; Piazuelo, M Blanca; Yan, Fang; Barry, Daniel P; Sierra, Johanna Carolina; Delgado, Alberto G; Hill, Salisha; Casero, Robert A; Bravo, Luis E; Dominguez, Ricardo L; Correa, Pelayo; Polk, D Brent; Washington, M Kay; Rose, Kristie L; Schey, Kevin L; Morgan, Douglas R; Peek, Richard M; Wilson, Keith T

2014-06-01

The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Protection of cadmium chloride induced DNA damage by Lamiaceae plants

PubMed Central

Thirugnanasampandan, Ramaraj; Jayakumar, Rajarajeswaran

2011-01-01

Objective To analyze the total phenolic content, DNA protecting and radical scavenging activity of ethanolic leaf extracts of three Lamiaceae plants, i.e. Anisomelos malabarica (A. malabarica), Leucas aspera (L. aspera) and Ocimum basilicum (O. basilicum). Methods The total polyphenols and flavonoids were analyzed in the ethanolic leaf extracts of the lamiaceae plants. To determine the DNA protecting activity, various concentrations of the plant extracts were prepared and treated on cultured HepG2 human lung cancer cells. The pretreated cells were exposed to H2O2 to induce DNA damage through oxidative stress. Comet assay was done and the tail length of individual comets was measured. Nitric oxide and superoxide anion scavenging activities of lamiaceae plants were analyzed. Results Among the three plant extracts, the highest amount of total phenolic content was found in O. basilicum (189.33 mg/g), whereas A. malabarica showed high levels of flavonoids (10.66 mg/g). O. basilicum also showed high levels of DNA protecting (85%) and radical scavenging activity. Conclusions The results of this study shows that bioactive phenols present in lamiaceae plants may prevent carcinogenesis through scavenging free radicals and inhibiting DNA damage. PMID:23569799

[Ubiquitin-proteasome system and sperm DNA repair: An update].

PubMed

Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

2016-09-01

The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1

PubMed Central

Dyson, Ossie F.; Pagano, Joseph S.

2017-01-01

ABSTRACT Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production. PMID:28724765

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

PubMed

Dyson, Ossie F; Pagano, Joseph S; Whitehurst, Christopher B

2017-10-01

Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae ; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production. Copyright © 2017 American Society for Microbiology.

DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

PubMed Central

Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

2011-01-01

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

Nedd4 Family Interacting Protein 1 (Ndfip1) Is Required for Ubiquitination and Nuclear Trafficking of BRCA1-associated ATM Activator 1 (BRAT1) during the DNA Damage Response*

PubMed Central

Low, Ley-Hian; Chow, Yuh-Lit; Li, Yijia; Goh, Choo-Peng; Putz, Ulrich; Silke, John; Ouchi, Toru; Howitt, Jason; Tan, Seong-Seng

2015-01-01

During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1. PMID:25631046

Effects of low dose pre-irradiation on hepatic damage and genetic material damage caused by cyclophosphamide.

PubMed

Yu, H-S; Song, A-Q; Liu, N; Wang, H

2014-01-01

Cyclophosphamide (CTX) can attack tumour cells, but can also damage the other cells and microstructures of an organism at different levels, such as haematopoietic cells, liver cells, peripheral lymphocyte DNA, and genetic materials. Low dose radiation (LDR) can induce general adaptation reaction. In this study, we explore the effects of low dose radiation on hepatic damage and genetic material damage caused by CTX. Mice were implanted subcutaneously with S180 cells in the left groin (control group excluded). On days 8 and 11, mice of the LDR and LDR+CTX groups were given 75 mGy of whole-body γ-irradiation; whereas mice of the CTX and LDR+CTX groups were injected intraperitoneally with 3.0 mg of CTX. All mice were sacrificed on day 13. DNA damage of the peripheral lymphocytes, alanine aminotransferase (ALT) activity, total protein (TP), albumin (ALB) of the plasma, malonyl-dialdheyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity of the hepatic homogenate, and micronucleus frequency (MNF) of polychromatoerythrocytes in the bone marrow were analysed. The control group had the lowest MDA content and the highest SOD and GSH-PX activity, whereas the CTX group had the highest MDA content and the lowest SOD and GSH-PX activity. Compared with the CTX group, the MDA content decreased significantly (p < 0.01) and the SOD and GSH-PX activity increased significantly (p < 0.05) in the LDR+CTX group. TP and ALB in control group were higher than that of the other groups. Compared with the sham-irradiated group, TP and ALB in the LDR group elevated significantly (p < 0.05). The control group had the lightest DNA damage, whereas the CTX group had the severest. DNA damage in LDR+CTX group was much lighter compared with that of the CTX group (p < 0.05). MNF in the CTX group increased significantly compared with the control and the sham-irradiated groups (p < 0.01). Compared with the CTX group, MNF in LDR+CTX group had a tendency of decline, but without statistical significance (p > 0.05). Pre-chemotherapeutic LDR can induce the activities of anti-oxidative enzymes and promote the elimination of free radicles to alleviate the damaging effects of oxidative stress to hepatic tissue caused by high-dose CTX. At the same time, LDR has no obvious effect on the ALT activity of plasma, but may have protective effect on the protein synthesis function of the liver. High-dose CTX chemotherapy can cause DNA damage of peripheral lymphocytes; however, LDR before chemotherapy may have certain protective effect on DNA damage. Moreover, CTX has potent mutagenic effect; however, LDR may have no protective effect against the genetic toxicity of CTX chemotherapy.

DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells.

PubMed

Lim, Hui Kheng; Gurung, Resham Lal; Hande, M Prakash

2017-12-01

Silver nanoparticles (Ag-np) were reported to be toxic to eukaryotic cells. These potentially detrimental effects of Ag-np can be advantageous in experimental therapeutics. They are currently being employed to enhance the therapeutic efficacy of cancer drugs. In this study, we demonstrate that Ag-np treatment trigger the activation of DNA-PKcs and JNK pathway at selected doses, presumably as a physiologic response to DNA damage and repair in normal and malignant cells. Ag-np altered the telomere dynamics by disrupting the shelterin complex located at the telomeres and telomere lengths. The genotoxic effect of Ag-np was not restricted to telomeres but the entire genome as Ag-np induced γ-H2AX foci formation, an indicator of global DNA damage. Inhibition of DNA-PKcs activity sensitised the cancer cells towards the cytotoxicity of Ag-np and substantiated the damaging effect of Ag-np at telomeres in human cancer cells. Abrogation of JNK mediated DNA repair and extensive damage of telomeres led to greater cell death following Ag-np treatment in DNA-PKcs inhibited cancer cells. Collectively, this study suggests that improved anti-proliferative and cytotoxic effects of Ag-np treatment in cancer cells can be achieved by the inhibition of DNA-PKcs. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro effect of the antimalarial drug proguanil hydrochloride on viability and DNA damage in human peripheral blood lymphocytes.

PubMed

Gajski, Goran; Dinter, Domagoj; Garaj-Vrhovac, Vera

2010-11-01

This study aimed to evaluate the effect of proguanil, a chemical substance used for treatment and prevention of malaria on viability and DNA integrity in human lymphocytes in vitro. Two different concentrations of proguanil obtained from the plasma concentrations were used: 130ng/ml used for prophylactic treatment and 520ng/ml used in treatment of malaria. Testing was done with and without metabolic activation. Viability of lymphocytes decreased in time and dose dependent manner. Comet assay parameters showed similar effects, indicating that some damage to DNA molecule can occur. Frequency of sister chromatid exchanges did not show significant deviation from the control samples. As for the proliferation kinetics no significant changes were noticed. Since majority of DNA damaging effect is induced after metabolic activation it is to be concluded that activity of proguanil is dependent upon the active metabolite cycloguanil and that monitoring should be conducted especially among frequent travellers. Copyright © 2010 Elsevier B.V. All rights reserved.

Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

PubMed Central

Foster, Patricia L.; Lee, Heewook; Popodi, Ellen; Townes, Jesse P.; Tang, Haixu

2015-01-01

A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10−3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold. PMID:26460006

Ebselen attenuates oxidative DNA damage and enhances its repair activity in the thalamus after focal cortical infarction in hypertensive rats.

PubMed

He, Meixia; Xing, Shihui; Yang, Bo; Zhao, Liqun; Hua, Haiying; Liang, Zhijian; Zhou, Wenliang; Zeng, Jinsheng; Pei, Zhong

2007-11-21

Oxidative DNA damage has been proposed to be a major contributor to focal cerebral ischemic injury. However, little is known about the role of oxidative DNA damage in remote damage secondary to the primary infarction. In the present study, we investigated oxidative damage within the ventroposterior nucleus (VPN) after distal middle cerebral artery occlusion (MCAO) in hypertensive rats. We also examined the possible protective effect of ebselen, one glutathione peroxidase mimic, on delayed degeneration in the VPN after distal MCAO. Neuronal damage in the ipsilateral VPN was examined by Nissl staining. Oxidative DNA damage and base repair enzyme activity were assessed by analyzing immunoreactivity of 8-hydroxy-2'-deoxyguanosine (8-ohdG) and 8-oxoguanine DNA glycosylase (OGG1), respectively. The number of intact neurons in the ipsilateral VPN decreased by 52% compared to the contralateral side in ischemia group 2 weeks after distal cerebral cortical infarction. The immunoreactivity of 8-ohdG significantly increased while OGG1 immunoreactivity significantly decreased in the ipsilateral VPN 2 weeks after distal cortical infarction (all p<0.01). Compared with vehicle treatment, ebselen significantly attenuated the neuron loss, ameliorated ischemia-induced increase in 8-ohdG level as well as decrease in OGG1 level within the ipsilateral VPN (all p<0.01). OGG1 was further demonstrated to mainly express in neurons. These findings strongly suggest that oxidative DNA damage may be involved in the delayed neuronal death in the VPN region following distal MCAO. Furthermore, ebselen protects against the delayed damage in the VPN when given at 24 h following distal MCAO.

Cell-free chromatin from dying cancer cells integrate into genomes of bystander healthy cells to induce DNA damage and inflammation

PubMed Central

Mittra, Indraneel; Samant, Urmila; Sharma, Suvarna; Raghuram, Gorantla V; Saha, Tannistha; Tidke, Pritishkumar; Pancholi, Namrata; Gupta, Deepika; Prasannan, Preeti; Gaikwad, Ashwini; Gardi, Nilesh; Chaubal, Rohan; Upadhyay, Pawan; Pal, Kavita; Rane, Bhagyeshri; Shaikh, Alfina; Salunkhe, Sameer; Dutt, Shilpee; Mishra, Pradyumna K; Khare, Naveen K; Nair, Naveen K; Dutt, Amit

2017-01-01

Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell–cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities. PMID:28580170

Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant

NASA Astrophysics Data System (ADS)

Moe, Elin; Rollo, Filipe; Silveira, Célia M.; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

2018-01-01

Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex.

Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant.

PubMed

Moe, Elin; Rollo, Filipe; Silveira, Célia M; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

2018-01-05

Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII 2 ). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII 2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII 2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex. Copyright © 2017 Elsevier B.V. All rights reserved.

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

PubMed Central

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

2015-01-01

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

Transcriptional activation of short interspersed elements by DNA-damaging agents.

PubMed

Rudin, C M; Thompson, C B

2001-01-01

Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

PubMed

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

DNA damage protective effect of honey-sweetened cashew apple nectar in Drosophila melanogaster

PubMed Central

da Silva, Robson Alves; Dihl, Rafael Rodrigues; Dias, Lucas Pinheiro; Costa, Maiane Papke; de Abreu, Bianca Regina Ribas; Cunha, Kênya Silva; Lehmann, Mauricio

2016-01-01

Abstract Fruits and derivatives, such as juices, are complex mixtures of chemicals, some of which may have mutagenic and/or carcinogenic potential, while others may have antimutagenic and/or anticancer activities. The modulating effects of honey-sweetened cashew apple nectar (HSCAN), on somatic mutation and recombination induced by ethyl methanesulfonate (EMS) and mitomycin C (MMC) were evaluated with the wing spot test in Drosophila melanogaster using co- and post-treatment protocols. Additionally, the antimutagenic activity of two HSCAN components, cashew apple pulp and honey, in MMC-induced DNA damage was also investigated. HSCAN reduced the mutagenic activity of both EMS and MMC in the co-treatment protocol, but had a co-mutagenic effect when post-administered. Similar results were also observed with honey on MMC mutagenic activity. Cashew apple pulp was effective in exerting protective or enhancing effects on the MMC mutagenicity, depending on the administration protocol and concentration used. Overall, these results indicate that HSCAN, cashew apple and honey seem capable of modulating not only the events that precede the induced DNA damages, but also the Drosophila DNA repair processes involved in the correction of EMS and MMC-induced damages. PMID:27560988

Electrochemical detection of DNA damage induced by acrylamide and its metabolite at the graphene-ionic liquid-Nafion modified pyrolytic graphite electrode.

PubMed

Qiu, Yanyan; Qu, Xiangjin; Dong, Jing; Ai, Shiyun; Han, Ruixia

2011-06-15

A new electrochemical biosensor for directly detecting DNA damage induced by acrylamide (AA) and its metabolite was presented in this work. The graphene-ionic liquid-Nafion modified pyrolytic graphite electrode (PGE) was prepared, and then horseradish peroxidase (HRP) and natural double-stranded DNA were alternately assembled on the modified electrode by the layer-by-layer method. The PGE/graphene-ionic liquid-Nafion and the construction of the (HRP/DNA)(n) film were characterized by electrochemical impedance spectroscopy. With the guanine signal in DNA as an indicator, the damage of DNA was detected by differential pulse voltammetry after PGE/graphene-ionic liquid-Nafion/(HRP/DNA)(n) was incubated in AA solution or AA+H(2)O(2) solution at 37°C. This method provides a new model to mimic and directly detect DNA damage induced by chemical pollutants and their metabolites in vitro. The results indicated that, in the presence of H(2)O(2), HRP was activated and catalyzed the transformation of AA to glycidamide, which could form DNA adducts and induce more serious damage of DNA than AA. In order to further verify these results, UV-vis spectrophotometry was also used to investigate DNA damage induced by AA and its metabolites in solution and the similar results were obtained. Copyright © 2011 Elsevier B.V. All rights reserved.

DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro

PubMed Central

Witte, Otto W.; Grosskreutz, Julian

2017-01-01

Mutations in the human Cu/Zn superoxide dismutase type-1 (hSOD1) gene are common in familial amyotrophic lateral sclerosis (fALS). The pathophysiology has been linked to, e.g., organelle dysfunction, RNA metabolism and oxidative DNA damage conferred by SOD1 malfunction. However, apart from metabolically evoked DNA oxidation, it is unclear whether severe genotoxicity including DNA single-strand breaks (SSBs) and double-strand breaks (DSBs), originates from loss of function of nuclear SOD1 enzyme. Factors that endogenously interfere with DNA integrity and repair complexes in hSOD1-mediated fALS remain similarly unexplored. In this regard, uncontrolled activation of transposable elements (TEs) might contribute to DNA disintegration and neurodegeneration. The aim of this study was to elucidate the role of the fALS-causing hSOD1G93A mutation in the generation of severe DNA damage beyond well-characterized DNA base oxidation. Therefore, DNA damage was assessed in spinal tissue of hSOD1G93A-overexpressing mice and in corresponding motor neuron-enriched cell cultures in vitro. Overexpression of the hSOD1G93A locus did not change the threshold for severe DNA damage per se. We found that levels of SSBs and DSBs were unaltered between hSOD1G93A and control conditions, as demonstrated in post-mitotic motor neurons and in astrocytes susceptible to replication-dependent DNA breakage. Analogously, parameters indicative of DNA damage response processes were not activated in vivo or in vitro. Evidence for a mutation-related elevation in TE activation was not detected, in accordance with the absence of TAR DNA binding protein 43 (TDP-43) proteinopathy in terms of cytoplasmic mislocation or nuclear loss, as nuclear TDP-43 is supposed to silence TEs physiologically. Conclusively, the superoxide dismutase function of SOD1 might not be required to preserve DNA integrity in motor neurons, at least when the function of TDP-43 is unaltered. Our data establish a foundation for further investigations addressing functional TDP-43 interaction with ALS-relevant genetic mutations. PMID:28832631

Mitogen-activated protein kinase signal transduction and DNA repair network are involved in aluminum-induced DNA damage and adaptive response in root cells of Allium cepa L.

PubMed Central

Panda, Brahma B.; Achary, V. Mohan M.

2014-01-01

In the current study, we studied the role of signal transduction in aluminum (Al3+)-induced DNA damage and adaptive response in root cells of Allium cepa L. The root cells in planta were treated with Al3+ (800 μM) for 3 h without or with 2 h pre-treatment of inhibitors of mitogen-activated protein kinase (MAPK), and protein phosphatase. Also, root cells in planta were conditioned with Al3+ (10 μM) for 2 h and then subjected to genotoxic challenge of ethyl methane sulfonate (EMS; 5 mM) for 3 h without or with the pre-treatment of the aforementioned inhibitors as well as the inhibitors of translation, transcription, DNA replication and repair. At the end of treatments, roots cells were assayed for cell death and/or DNA damage. The results revealed that Al3+ (800 μM)-induced significant DNA damage and cell death. On the other hand, conditioning with low dose of Al3+ induced adaptive response conferring protection of root cells from genotoxic stress caused by EMS-challenge. Pre-treatment of roots cells with the chosen inhibitors prior to Al3+-conditioning prevented or reduced the adaptive response to EMS genotoxicity. The results of this study suggested the involvement of MAPK and DNA repair network underlying Al-induced DNA damage and adaptive response to genotoxic stress in root cells of A. cepa. PMID:24926302

p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

PubMed Central

Hwang, Byung Joon; Toering, Stephanie; Francke, Uta; Chu, Gilbert

1998-01-01

A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin. PMID:9632823

Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated.

PubMed

Tognetti, Silvia; Riera, Alberto; Speck, Christian

2015-03-01

A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2-7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2-7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

PubMed

Tran, Thai Q; Ishak Gabra, Mari B; Lowman, Xazmin H; Yang, Ying; Reid, Michael A; Pan, Min; O'Connor, Timothy R; Kong, Mei

2017-11-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes

PubMed Central

Tran, Thai Q.; Ishak Gabra, Mari B.; Lowman, Xazmin H.; Yang, Ying; Reid, Michael A.; Pan, Min; O’Connor, Timothy R.

2017-01-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer. PMID:29107960

The G2 block induced by DNA damage: a caffeine-resistant component independent of Cdc25C, MPM-2 phosphorylation, and H1 kinase activity.

PubMed

Barratt, R A; Kao, G; McKenna, W G; Kuang, J; Muschel, R J

1998-06-15

Treatment of cells with agents that cause DNA damage often results in a delay in G2. There is convincing evidence showing that inhibition of p34cdc2 kinase activation is involved in the DNA damage-induced G2 delay. In this study, we have demonstrated the existence of an additional pathway, independent of the p34cdc2 kinase activation pathway, that leads to a G2 arrest in etoposide-treated cells. Both the X-ray-induced and the etoposide-induced G2 arrest were associated with inhibition of the p34cdc2 H1 kinase activation pathway as judged by p34cdc2 H1 kinase activity and phosphorylation of cdc25C. Caffeine treatment restored these activities after either of the treatments. However, the etoposide-treated cells did not resume cycling, revealing the presence of an alternative pathway leading to a G2 arrest. To explore the possibility that this additional pathway involved phosphorylation of the MPM-2 epitope that is shared by a large family of mitotic phosphoproteins, we monitored the phosphorylation status of the MPM-2 epitope after DNA damage and after treatment with caffeine. Phosphorylation of the MPM-2 epitope was depressed in both X-ray and etoposide-treated cells, and the depression was reversed by caffeine in both cases. The results indicate that the pathway affecting MPM-2 epitope phosphorylation is involved in the G2 delay caused by DNA damage. However, it is not part of the caffeine-insensitive pathway leading to a G2 block seen in etoposide-treated cells.

Dietary spices protect against hydrogen peroxide-induced DNA damage and inhibit nicotine-induced cancer cell migration.

PubMed

Jayakumar, R; Kanthimathi, M S

2012-10-01

Spices are rich sources of antioxidants due to the presence of phenols and flavonoids. In this study, the DNA protecting activity and inhibition of nicotine-induced cancer cell migration of 9 spices were analysed. Murine fibroblasts (3T3-L1) and human breast cancer (MCF-7) cells were pre-treated with spice extracts and then exposed to H₂O₂ and nicotine. The comet assay was used to analyse the DNA damage. Among the 9 spices, ginger, at 50 μg/ml protected against 68% of DNA damage in 3T3-L1 cells. Caraway, cumin and fennel showed statistically significant (p<0.05) DNA protecting activity. Treatment of MCF-7 cells with nicotine induced cell migration, whereas pre-treatment with spices reduced this migration. Pepper, long pepper and ginger exhibited a high rate of inhibition of cell migration. The results of this study prove that spices protect DNA and inhibit cancer cell migration. Copyright © 2012 Elsevier Ltd. All rights reserved.

Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria

PubMed Central

Peng, Linyuan; Tang, Xiaolong; Meng, Fanbiao; Ao, Ying; Zhou, Mingyan; Wang, Ming; Cao, Xinyue; Qin, Baoming; Wang, Zimei; Zhou, Zhongjun; Wang, Guangming; Gao, Zhengliang; Xu, Jun

2018-01-01

DNA damage accumulates with age (Lombard et al., 2005). However, whether and how robust DNA repair machinery promotes longevity is elusive. Here, we demonstrate that ATM-centered DNA damage response (DDR) progressively declines with senescence and age, while low dose of chloroquine (CQ) activates ATM, promotes DNA damage clearance, rescues age-related metabolic shift, and prolongs replicative lifespan. Molecularly, ATM phosphorylates SIRT6 deacetylase and thus prevents MDM2-mediated ubiquitination and proteasomal degradation. Extra copies of Sirt6 extend lifespan in Atm-/- mice, with restored metabolic homeostasis. Moreover, the treatment with CQ remarkably extends lifespan of Caenorhabditis elegans, but not the ATM-1 mutants. In a progeria mouse model with low DNA repair capacity, long-term administration of CQ ameliorates premature aging features and extends lifespan. Thus, our data highlights a pro-longevity role of ATM, for the first time establishing direct causal links between robust DNA repair machinery and longevity, and providing therapeutic strategy for progeria and age-related metabolic diseases. PMID:29717979

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kao, Shyan-Yuan, E-mail: shyan-yuan_kao@meei.harvard.edu

Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant.more » Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.« less

Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria.

PubMed

Qian, Minxian; Liu, Zuojun; Peng, Linyuan; Tang, Xiaolong; Meng, Fanbiao; Ao, Ying; Zhou, Mingyan; Wang, Ming; Cao, Xinyue; Qin, Baoming; Wang, Zimei; Zhou, Zhongjun; Wang, Guangming; Gao, Zhengliang; Xu, Jun; Liu, Baohua

2018-05-02

DNA damage accumulates with age (Lombard et al., 2005). However, whether and how robust DNA repair machinery promotes longevity is elusive. Here, we demonstrate that ATM-centered DNA damage response (DDR) progressively declines with senescence and age, while low dose of chloroquine (CQ) activates ATM, promotes DNA damage clearance, rescues age-related metabolic shift, and prolongs replicative lifespan. Molecularly, ATM phosphorylates SIRT6 deacetylase and thus prevents MDM2-mediated ubiquitination and proteasomal degradation. Extra copies of Sirt6 extend lifespan in Atm-/- mice, with restored metabolic homeostasis. Moreover, the treatment with CQ remarkably extends lifespan of Caenorhabditis elegans , but not the ATM-1 mutants. In a progeria mouse model with low DNA repair capacity, long-term administration of CQ ameliorates premature aging features and extends lifespan. Thus, our data highlights a pro-longevity role of ATM, for the first time establishing direct causal links between robust DNA repair machinery and longevity, and providing therapeutic strategy for progeria and age-related metabolic diseases. © 2018, Qian et al.

APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallace, Bret D.; Berman, Zachary; Mueller, Geoffrey A.

The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structuremore » and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3'-5' exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.« less

Oxidative DNA damage and its repair in rat spleen following subchronic exposure to aniline

PubMed Central

Ma, Huaxian; Wang, Jianling; Abdel-Rahman, Sherif Z.; Boor, Paul J.; Khan, M. Firoze

2008-01-01

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Splenotoxicity of aniline is associated with iron overload and generation of reactive oxygen species (ROS) which can cause oxidative damage to DNA, proteins and lipids (oxidative stress). 8-Hydroxy-2’-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA lesions resulting from ROS, and 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase/lyase enzyme, plays a key role in the removal of 8-OHdG adducts. This study focused on examining DNA damage (8-OHdG) and repair (OGG1) in the spleen in an experimental condition preceding a tumorigenic response. To achieve that, male Sprague-Dawley rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. Aniline treatment led to a significant increase in splenic oxidative DNA damage, manifested as a 2.8-fold increase in 8-OHdG levels. DNA repair activity, measured as OGG1 base excision repair (BER) activity, increased by ~1.3 fold in the nuclear protein extracts (NE) and ~1.2 fold in the mitochondrial protein extracts (ME) of spleens from aniline-treated rats as compared to the controls. Real-time PCR analysis for OGG1 mRNA expression in the spleen revealed a 2-fold increase in expression in aniline-treated rats than the controls. Likewise, OGG1 protein expression in the NEs of spleens from aniline-treated rats was ~1.5 fold higher, whereas in the MEs it was ~1.3 fold higher than the controls. Aniline treatment also led to stronger immunostaining for both 8-OHdG and OGG1 in the spleens, confined to the red pulp areas. It is thus evident from our studies that aniline-induced oxidative stress is associated with increased oxidative DNA damage. The BER pathway was also activated, but not enough to prevent the accumulation of oxidative DNA damage (8-OHdG). Accumulation of mutagenic oxidative DNA lesions in the spleen following exposure to aniline could play a critical role in the tumorigenic process. PMID:18793663

A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage

PubMed Central

2017-01-01

The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes autoproteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway. PMID:28826208

Choline deficiency increases lymphocyte apoptosis and DNA damage in humans2,3

PubMed Central

da Costa, Kerry-Ann; Niculescu, Mihai D; Craciunescu, Corneliu N; Fischer, Leslie M; Zeisel, Steven H

2008-01-01

Background: Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans. Objective: The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans. Design: Fifty-one men and women aged 18–70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138–550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase–mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays. Results: All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet. Conclusions: A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dys-function also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected. PMID:16825685

Assessment of DNA damage in a group of professional dancers during a 10-month dancing season.

PubMed

Esteves, Filipa; Teixeira, Eduardo; Amorim, Tânia; Costa, Carla; Pereira, Cristiana; Fraga, Sónia; De Andrade, Vanessa Moraes; Teixeira, João Paulo; Costa, Solange

2017-01-01

Despite the numerous health benefits of physical activity, some studies reported that increased intensity and duration may induce oxidative stress in several cellular components including DNA. The aim of this study was to assess the level of basal DNA damage as well as oxidative DNA damage in a group of professional dancers before and after a 10-month dancing season. A group of individuals from general population was also assessed as a control. The alkaline version of the comet assay was the method selected to measure both basal DNA damage and oxidative stress, since this method quantifies both endpoints. In order to measure oxidative stress, the comet assay was coupled with a lesion-specific endonuclease (formamidopyrimidine glycosylase) to detect oxidized purines. The levels of oxidative DNA damage in dancers were significantly increased after the dancing season. Pre-season levels of oxidative DNA damage were lower in dancers than those obtained from the general population, suggesting an adaptation of antioxidant system in dancers. Results of the present biomonitoring study indicate the need for more effective measures to protect ballet dancers from potentially occupational health risks related to regular intensive physical exercise.

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.

The RNA Splicing Response to DNA Damage

PubMed Central

Shkreta, Lulzim; Chabot, Benoit

2015-01-01

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

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

PubMed

Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

2017-08-01

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G 2 -M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR . ©2017 American Association for Cancer Research.

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway.

PubMed

Nakada, Daisuke; Hirano, Yukinori; Sugimoto, Katsunori

2004-11-01

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

PubMed Central

Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

2013-01-01

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways

PubMed Central

Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

2006-01-01

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms. PMID:16710445

Epigenetic telomere protection by Drosophila DNA damage response pathways.

PubMed

Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

2006-05-01

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

DNA damage in children and adolescents with cardiovascular disease risk factors.

PubMed

Kliemann, Mariele; Prá, Daniel; Müller, Luiza L; Hermes, Liziane; Horta, Jorge A; Reckziegel, Miriam B; Burgos, Miria S; Maluf, Sharbel W; Franke, Silvia I R; Silva, Juliana da

2012-09-01

The risk of developing cardiovascular disease (CVD) is related to lifestyle (e.g. diet, physical activity and smoking) as well as to genetic factors. This study aimed at evaluating the association between CVD risk factors and DNA damage levels in children and adolescents. Anthropometry, diet and serum CVD risk factors were evaluated by standard procedures. DNA damage levels were accessed by the comet assay (Single cell gel electrophoresis; SCGE) and cytokinesis-blocked micronucleus (CBMN) assays in leukocytes. A total of 34 children and adolescents selected from a population sample were divided into three groups according to their level of CVD risk. Moderate and high CVD risk subjects showed significantly higher body fat and serum CVD risk markers than low risk subjects (P<0.05). High risk subjects also showed a significant increase in DNA damage, which was higher than that provided by low and moderate risk subjects according to SCGE, but not according to the CBMN assay. Vitamin C intake was inversely correlated with DNA damage by SCGE, and micronucleus (MN) was inversely correlated with folate intake. The present results indicate an increase in DNA damage that may be a consequence of oxidative stress in young individuals with risk factors for CVD, indicating that the DNA damage level can aid in evaluating the risk of CVD.

ATM-Dependent Phosphorylation of MEF2D Promotes Neuronal Survival after DNA Damage

PubMed Central

Chan, Shing Fai; Sances, Sam; Brill, Laurence M.; Okamoto, Shu-ichi; Zaidi, Rameez; McKercher, Scott R.; Akhtar, Mohd W.; Nakanishi, Nobuki

2014-01-01

Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM–MEF2D pathway may contribute to neurodegeneration in AT. PMID:24672010

Cytotoxic and DNA-damaging properties of glyphosate and Roundup in human-derived buccal epithelial cells.

PubMed

Koller, Verena J; Fürhacker, Maria; Nersesyan, Armen; Mišík, Miroslav; Eisenbauer, Maria; Knasmueller, Siegfried

2012-05-01

Glyphosate (G) is the largest selling herbicide worldwide; the most common formulations (Roundup, R) contain polyoxyethyleneamine as main surfactant. Recent findings indicate that G exposure may cause DNA damage and cancer in humans. Aim of this investigation was to study the cytotoxic and genotoxic properties of G and R (UltraMax) in a buccal epithelial cell line (TR146), as workers are exposed via inhalation to the herbicide. R induced acute cytotoxic effects at concentrations > 40 mg/l after 20 min, which were due to membrane damage and impairment of mitochondrial functions. With G, increased release of extracellular lactate dehydrogenase indicative for membrane damage was observed at doses > 80 mg/l. Both G and R induced DNA migration in single-cell gel electrophoresis assays at doses > 20 mg/l. Furthermore, an increase of nuclear aberrations that reflect DNA damage was observed. The frequencies of micronuclei and nuclear buds were elevated after 20-min exposure to 10-20 mg/l, while nucleoplasmatic bridges were only enhanced by R at the highest dose (20 mg/l). R was under all conditions more active than its active principle (G). Comparisons with results of earlier studies with lymphocytes and cells from internal organs indicate that epithelial cells are more susceptible to the cytotoxic and DNA-damaging properties of the herbicide and its formulation. Since we found genotoxic effects after short exposure to concentrations that correspond to a 450-fold dilution of spraying used in agriculture, our findings indicate that inhalation may cause DNA damage in exposed individuals.

Balancing repair and tolerance of DNA damage caused by alkylating agents.

PubMed

Fu, Dragony; Calvo, Jennifer A; Samson, Leona D

2012-01-12

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity.

SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

PubMed Central

Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

2013-01-01

Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for an organism's favorable response to alkylating agents. Furthermore, an individual's response to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity. PMID:22237395

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krüger, Katharina; Ziegler, Verena; Hartmann, Christina

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.more » 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. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of CisPt-triggered DDR leads to cytoprotection. • Lovastatin attenuates CisPt-induced kidney DNA damage and apoptosis in vivo.« less

HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production.

PubMed

El-Amine, Rawan; Germini, Diego; Zakharova, Vlada V; Tsfasman, Tatyana; Sheval, Eugene V; Louzada, Ruy A N; Dupuy, Corinne; Bilhou-Nabera, Chrystèle; Hamade, Aline; Najjar, Fadia; Oksenhendler, Eric; Lipinski, Marс; Chernyak, Boris V; Vassetzky, Yegor S

2018-05-01

Human immunodeficiency virus (HIV) infection is associated with B-cell malignancies in patients though HIV-1 is not able to infect B-cells. The rate of B-cell lymphomas in HIV-infected individuals remains high even under the combined antiretroviral therapy (cART) that reconstitutes the immune function. Thus, the contribution of HIV-1 to B-cell oncogenesis remains enigmatic. HIV-1 induces oxidative stress and DNA damage in infected cells via multiple mechanisms, including viral Tat protein. We have detected elevated levels of reactive oxygen species (ROS) and DNA damage in B-cells of HIV-infected individuals. As Tat is present in blood of infected individuals and is able to transduce cells, we hypothesized that it could induce oxidative DNA damage in B-cells promoting genetic instability and malignant transformation. Indeed, incubation of B-cells isolated from healthy donors with purified Tat protein led to oxidative stress, a decrease in the glutathione (GSH) levels, DNA damage and appearance of chromosomal aberrations. The effects of Tat relied on its transcriptional activity and were mediated by NF-κB activation. Tat stimulated oxidative stress in B-cells mostly via mitochondrial ROS production which depended on the reverse electron flow in Complex I of respiratory chain. We propose that Tat-induced oxidative stress, DNA damage and chromosomal aberrations are novel oncogenic factors favoring B-cell lymphomas in HIV-1 infected individuals. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

A Protective Mechanism of Visible Red Light in Normal Human Dermal Fibroblasts: Enhancement of GADD45A-Mediated DNA Repair Activity.

PubMed

Kim, Yeo Jin; Kim, Hyoung-June; Kim, Hye Lim; Kim, Hyo Jeong; Kim, Hyun Soo; Lee, Tae Ryong; Shin, Dong Wook; Seo, Young Rok

2017-02-01

The phototherapeutic effects of visible red light on skin have been extensively investigated, but the underlying biological mechanisms remain poorly understood. We aimed to elucidate the protective mechanism of visible red light in terms of DNA repair of UV-induced oxidative damage in normal human dermal fibroblasts. The protective effect of visible red light on UV-induced DNA damage was identified by several assays in both two-dimensional and three-dimensional cell culture systems. With regard to the protective mechanism of visible red light, our data showed alterations in base excision repair mediated by growth arrest and DNA damage inducible, alpha (GADD45A). We also observed an enhancement of the physical activity of GADD45A and apurinic/apyrimidinic endonuclease 1 (APE1) by visible red light. Moreover, UV-induced DNA damages were diminished by visible red light in an APE1-dependent manner. On the basis of the decrease in GADD45A-APE1 interaction in the activating transcription factor-2 (ATF2)-knockdown system, we suggest a role for ATF2 modulation in GADD45A-mediated DNA repair upon visible red light exposure. Thus, the enhancement of GADD45A-mediated base excision repair modulated by ATF2 might be a potential protective mechanism of visible red light. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Corrupting the DNA damage response: a critical role for Rad52 in tumor cell survival.

PubMed

Lieberman, Rachel; You, Ming

2017-07-15

The DNA damage response enables cells to survive, maintain genome integrity, and to safeguard the transmission of high-fidelity genetic information. Upon sensing DNA damage, cells respond by activating this multi-faceted DNA damage response leading to restoration of the cell, senescence, programmed cell death, or genomic instability if the cell survives without proper repair. However, unlike normal cells, cancer cells maintain a marked level of genomic instability. Because of this enhanced propensity to accumulate DNA damage, tumor cells rely on homologous recombination repair as a means of protection from the lethal effect of both spontaneous and therapy-induced double-strand breaks (DSBs) in DNA. Thus, modulation of DNA repair pathways have important consequences for genomic instability within tumor cell biology and viability maintenance under high genotoxic stress. Efforts are underway to manipulate specific components of the DNA damage response in order to selectively induce tumor cell death by augmenting genomic instability past a viable threshold. New evidence suggests that RAD52, a component of the homologous recombination pathway, is important for the maintenance of tumor genome integrity. This review highlights recent reports indicating that reducing homologous recombination through inhibition of RAD52 may represent an important focus for cancer therapy and the specific efforts that are already demonstrating potential.

Magnesium Supplementation Diminishes Peripheral Blood Lymphocyte DNA Oxidative Damage in Athletes and Sedentary Young Man

PubMed Central

Petrović, Jelena; Stanić, Dušanka; Dmitrašinović, Gordana; Plećaš-Solarović, Bosiljka; Ignjatović, Svetlana; Batinić, Bojan; Popović, Dejana

2016-01-01

Sedentary lifestyle is highly associated with increased risk of cardiovascular disease, obesity, and type 2 diabetes. It is known that regular physical activity has positive effects on health; however several studies have shown that acute and strenuous exercise can induce oxidative stress and lead to DNA damage. As magnesium is essential in maintaining DNA integrity, the aim of this study was to determine whether four-week-long magnesium supplementation in students with sedentary lifestyle and rugby players could prevent or diminish impairment of DNA. By using the comet assay, our study demonstrated that the number of peripheral blood lymphocytes (PBL) with basal endogenous DNA damage is significantly higher in rugby players compared to students with sedentary lifestyle. On the other hand, magnesium supplementation significantly decreased the number of cells with high DNA damage, in the presence of exogenous H2O2, in PBL from both students and rugby players, and markedly reduced the number of cells with medium DNA damage in rugby players compared to corresponding control nonsupplemented group. Accordingly, the results of our study suggest that four-week-long magnesium supplementation has marked effects in protecting the DNA from oxidative damage in both rugby players and in young men with sedentary lifestyle. Clinical trial is registered at ANZCTR Trial Id: ACTRN12615001237572. PMID:27042258

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

S phase entry causes homocysteine-induced death while ataxia telangiectasia and Rad3 related protein functions anti-apoptotically to protect neurons.

PubMed

Ye, Weizhen; Blain, Stacy W

2010-08-01

A major phenotype seen in neurodegenerative disorders is the selective loss of neurons due to apoptotic death and evidence suggests that inappropriate re-activation of cell cycle proteins in post-mitotic neurons may be responsible. To investigate whether reactivation of the G1 cell cycle proteins and S phase entry was linked with apoptosis, we examined homocysteine-induced neuronal cell death in a rat cortical neuron tissue culture system. Hyperhomocysteinaemia is a physiological risk factor for a variety of neurodegenerative diseases, including Alzheimer's disease. We found that in response to homocysteine treatment, cyclin D1, and cyclin-dependent kinases 4 and 2 translocated to the nucleus, and p27 levels decreased. Both cyclin-dependent kinases 4 and 2 regained catalytic activity, the G1 gatekeeper retinoblastoma protein was phosphorylated and DNA synthesis was detected, suggesting transit into S phase. Double-labelling immunofluorescence showed a 95% co-localization of anti-bromodeoxyuridine labelling with apoptotic markers, demonstrating that those cells that entered S phase eventually died. Neurons could be protected from homocysteine-induced death by methods that inhibited G1 phase progression, including down-regulation of cyclin D1 expression, inhibition of cyclin-dependent kinases 4 or 2 activity by small molecule inhibitors, or use of the c-Abl kinase inhibitor, Gleevec, which blocked cyclin D and cyclin-dependent kinase 4 nuclear translocation. However, blocking cell cycle progression post G1, using DNA replication inhibitors, did not prevent apoptosis, suggesting that death was not preventable post the G1-S phase checkpoint. While homocysteine treatment caused DNA damage and activated the DNA damage response, its mechanism of action was distinct from that of more traditional DNA damaging agents, such as camptothecin, as it was p53-independent. Likewise, inhibition of the DNA damage sensors, ataxia-telangiectasia mutant and ataxia telangiectasia and Rad3 related proteins, did not rescue apoptosis and in fact exacerbated death, suggesting that the DNA damage response might normally function neuroprotectively to block S phase-dependent apoptosis induction. As cell cycle events appear to be maintained in vivo in affected neurons for weeks to years before apoptosis is observed, activation of the DNA damage response might be able to hold cell cycle-induced death in check.

DNA damage and oxidative stress induced by imidacloprid exposure in the earthworm Eisenia fetida.

PubMed

Wang, Juan; Wang, Jinhua; Wang, Guangchi; Zhu, Lusheng; Wang, Jun

2016-02-01

To investigate the soil ecological effect of imidacloprid, earthworm Eisenia fetida was exposed to various concentrations of imidacloprid (0.10, 0.50, and 1.00 mg kg(-1) soil) respectively after 7, 14, 21, and 28 d. The effect of imidacloprid on reactive oxygen species (ROS) generation, antioxidant enzymes activity [superoxide dismutase (SOD) and catalase (CAT), glutathione S-transferase enzyme (GST)], malondialdehyde (MDA) content and DNA damage of the E. fetida was investigated. Significant increase of the ROS level was observed. The SOD and GST activity were significantly induced at most exposure intervals. CAT activity was inhibited and reflected a dose-dependent relationship on days 7, 14 and 21. High MDA levels were observed and the olive tail moment (OTM) as well as the percentage of DNA in the comet tail (tail DNA%) in comet assay declined with increasing concentrations and exposure time after 7 d. Our results suggested that the sub-chronic exposure of imidacloprid caused DNA damage and lipid peroxidation (LPO) leading to antioxidant responses in earthworm E. fetida. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mytych, Jennifer, E-mail: jennifermytych@gmail.com; Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa; Wos, Izabela

Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we showmore » that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.« less

DNA Repair and the Accumulation of Oxidatively Damaged DNA Are Affected by Fruit Intake in Mice

PubMed Central

Croteau, Deborah L.; de Souza-Pinto, Nadja C.; Harboe, Charlotte; Keijzers, Guido; Zhang, Yongqing; Becker, Kevin; Sheng, Shan

2010-01-01

AGING is associated with elevated oxidative stress and DNA damage. To achieve healthy aging, we must begin to understand how diet affects cellular processes. We postulated that fruit-enriched diets might initiate a program of enhanced DNA repair and thereby improve genome integrity. C57Bl/6 J mice were fed for 14 weeks a control diet or a diet with 8% peach or nectarine extract. The activities of DNA repair enzymes, the level of DNA damage, and gene expression changes were measured. Our study showed that repair of various oxidative DNA lesions was more efficient in liver extracts derived from mice fed fruit-enriched diets. In support of these findings, gas chromatography–mass spectrometry analysis revealed that there was a decrease in the levels of formamidopyrimidines in peach-fed mice compared with the controls. Additionally, microarray analysis revealed that NTH1 was upregulated in peach-fed mice. Taken together, these results suggest that an increased intake of fruits might modulate the efficiency of DNA repair, resulting in altered levels of DNA damage. PMID:20847039

ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage

PubMed Central

Maya, Ruth; Balass, Moshe; Kim, Seong-Tae; Shkedy, Dganit; Leal, Juan-Fernando Martinez; Shifman, Ohad; Moas, Miri; Buschmann, Thomas; Ronai, Ze'ev; Shiloh, Yosef; Kastan, Michael B.; Katzir, Ephraim; Oren, Moshe

2001-01-01

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258–260 and 393–395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2. PMID:11331603

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

PubMed

Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

2018-02-01

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

Cytologic Effects of Air Force Chemicals

DTIC Science & Technology

1978-09-01

pCi/ml, 60 Ci/mmole), hydroxyurea (10- 2M) to suppress replicative DNA synthesis, and with or without 4-nitroquinoline-l-oxide (4NQO, a DNA-damaging...organ cultures. The tissues were minced in cold, buffered saline and then incubated with 3 H-thymidine, hydroxyurea and 4NQO to damage cellular DNA...incorporation under these conditions is taken as an indication of DNA repair activity, and incorporation of 3H-thymidine in the absence of hydroxyurea and

3′-Phosphoadenosine 5′-phosphosulfate synthase 1 (PAPSS1) knockdown sensitizes non-small cell lung cancer cells to DNA damaging agents

PubMed Central

Leung, Ada W. Y.; Dragowska, Wieslawa H.; Ricaurte, Daniel; Kwok, Brian; Mathew, Veena; Roosendaal, Jeroen; Ahluwalia, Amith; Warburton, Corinna; Laskin, Janessa J.; Stirling, Peter C.; Qadir, Mohammed A.; Bally, Marcel B.

2015-01-01

Standard treatment for advanced non-small cell lung cancer (NSCLC) with no known driver mutation is platinum-based chemotherapy, which has a response rate of only 30–33%. Through an siRNA screen, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate PAPS, was identified as a novel platinum-sensitizing target in NSCLC cells. PAPSS1 knockdown in combination with low-dose (IC10) cisplatin reduces clonogenicity of NSCLC cells by 98.7% (p < 0.001), increases DNA damage, and induces G1/S phase cell cycle arrest and apoptosis. PAPSS1 silencing also sensitized NSCLC cells to other DNA crosslinking agents, radiation, and topoisomerase I inhibitors, but not topoisomerase II inhibitors. Chemo-sensitization was not observed in normal epithelial cells. Knocking out the PAPSS1 homolog did not sensitize yeast to cisplatin, suggesting that sulfate bioavailability for amino acid synthesis is not the cause of sensitization to DNA damaging agents. Rather, sensitization may be due to sulfation reactions involved in blocking the action of DNA damaging agents, facilitating DNA repair, promoting cancer cell survival under therapeutic stress or reducing the bioavailability of DNA damaging agents. Our study demonstrates for the first time that PAPSS1 could be targeted to improve the activity of multiple anticancer agents used to treat NSCLC. PMID:26220590

Biomarkers of oxidative stress and DNA damage in agricultural workers: A pilot study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muniz, Juan F.; McCauley, Linda; Scherer, J.

Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A study of pesticide applicators and farmworkers was conducted to examine the relationship between organophosphate pesticide exposure and biomarkers of oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay), and serum was analyzed for lipid peroxides (i.e., malondialdehyde, MDA). Cellular damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farmworkers andmore » applicators (p < 0.001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farmworkers or applicators (respectively) than in controls. Serum MDA levels were 4.9 times and 24 times higher in farmworkers or applicators (respectively) than in controls. DNA damage (Comet assay) and oxidative DNA repair were significantly greater in lymphocytes from applicators and farmworkers when compared with controls. Markers of oxidative stress (i.e., increased reactive oxygen species and reduced glutathione levels) and DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that organophosphate pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and a number of health effects.« less

A new compound, withangulatin A, promotes type II DNA topoisomerase-mediated DNA damage.

PubMed

Juang, J K; Huang, H W; Chen, C M; Liu, H J

1989-03-31

Withangulatin A, a new compound with a known chemical structure and from the antitumor Chinese herb Physalis angulata L, was found to act on topoisomerase II to induce topoisomerase II-mediated DNA damage in vitro. It has two effective dosage ranges of approximate 0.5 and 20 microM, with about one-third the activity of 20 microM VM-26.

[The effect of UV-irradiation on telomerase activity and other stress-related proteins in human lens epithelial cells].

PubMed

Wu, Zhi-hong; Zhang, Jin-song

2005-05-01

To investigate the changes and the role of telomerase activity and other stress-related proteins in the process of UV-induced DNA damage and repair in human lens epithelial cells. Human lens epithelial cells were irradiated at UV-doses 0.0 (control group) and 0.5, 1.5, 2.5, 3.5, 5.0, 7.5, 10.0 mJ/cm(2) (treated 1-7 group). Telomerase activity was determined by Telomerase Repeat Amplification Protocol-Enzyme Linked Immunosorbent Assay (TRAP-ELISA), p53, growth arrest and DNA damage inducible (GADD45), proliferating cell nuclear antigen (PCNA) and p16 protein levels were analyzed by Western blotting. Telomerase activity in control group and treated 1-7 group showed increased tendency, the differences of telomerase activity in 8 groups were significantly (P < 0.01). The expression of p53, GADD45, PCNA, p16 proteins showed increased tendency in experimental group, comparing with the control group, there were significant difference (P < 0.01). During UV-induced DNA damage and repair in human lens epithelial cells, telomerase activity was upregulated and the expression of stress-related proteins levels was increased. Upregulated telomerase activity may play both a protective and a proliferative role in human lens epithelial cells. Increased stress-related proteins level is critic in UV-induced DNA damage and repair in human lens epithelial. Increased telomerase activity is associated with increased levels of the stress-related proteins.

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

PubMed

Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

2009-06-17

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

Omics Approaches for Identifying Physiological Adaptations to Genome Instability in Aging.

PubMed

Edifizi, Diletta; Schumacher, Björn

2017-11-04

DNA damage causally contributes to aging and age-related diseases. The declining functioning of tissues and organs during aging can lead to the increased risk of succumbing to aging-associated diseases. Congenital syndromes that are caused by heritable mutations in DNA repair pathways lead to cancer susceptibility and accelerated aging, thus underlining the importance of genome maintenance for withstanding aging. High-throughput mass-spectrometry-based approaches have recently contributed to identifying signalling response networks and gaining a more comprehensive understanding of the physiological adaptations occurring upon unrepaired DNA damage. The insulin-like signalling pathway has been implicated in a DNA damage response (DDR) network that includes epidermal growth factor (EGF)-, AMP-activated protein kinases (AMPK)- and the target of rapamycin (TOR)-like signalling pathways, which are known regulators of growth, metabolism, and stress responses. The same pathways, together with the autophagy-mediated proteostatic response and the decline in energy metabolism have also been found to be similarly regulated during natural aging, suggesting striking parallels in the physiological adaptation upon persistent DNA damage due to DNA repair defects and long-term low-level DNA damage accumulation occurring during natural aging. These insights will be an important starting point to study the interplay between signalling networks involved in progeroid syndromes that are caused by DNA repair deficiencies and to gain new understanding of the consequences of DNA damage in the aging process.

Omics Approaches for Identifying Physiological Adaptations to Genome Instability in Aging

PubMed Central

Edifizi, Diletta; Schumacher, Björn

2017-01-01

DNA damage causally contributes to aging and age-related diseases. The declining functioning of tissues and organs during aging can lead to the increased risk of succumbing to aging-associated diseases. Congenital syndromes that are caused by heritable mutations in DNA repair pathways lead to cancer susceptibility and accelerated aging, thus underlining the importance of genome maintenance for withstanding aging. High-throughput mass-spectrometry-based approaches have recently contributed to identifying signalling response networks and gaining a more comprehensive understanding of the physiological adaptations occurring upon unrepaired DNA damage. The insulin-like signalling pathway has been implicated in a DNA damage response (DDR) network that includes epidermal growth factor (EGF)-, AMP-activated protein kinases (AMPK)- and the target of rapamycin (TOR)-like signalling pathways, which are known regulators of growth, metabolism, and stress responses. The same pathways, together with the autophagy-mediated proteostatic response and the decline in energy metabolism have also been found to be similarly regulated during natural aging, suggesting striking parallels in the physiological adaptation upon persistent DNA damage due to DNA repair defects and long-term low-level DNA damage accumulation occurring during natural aging. These insights will be an important starting point to study the interplay between signalling networks involved in progeroid syndromes that are caused by DNA repair deficiencies and to gain new understanding of the consequences of DNA damage in the aging process. PMID:29113067

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.

DNA repair phenotype and dietary antioxidant supplementation.

PubMed

Guarnieri, Serena; Loft, Steffen; Riso, Patrizia; Porrini, Marisa; Risom, Lotte; Poulsen, Henrik E; Dragsted, Lars O; Møller, Peter

2008-05-01

Phytochemicals may protect cellular DNA by direct antioxidant effect or modulation of the DNA repair activity. We investigated the repair activity towards oxidised DNA in human mononuclear blood cells (MNBC) in two placebo-controlled antioxidant intervention studies as follows: (1) well-nourished subjects who ingested 600 g fruits and vegetables, or tablets containing the equivalent amount of vitamins and minerals, for 24 d; (2) poorly nourished male smokers who ingested 500 mg vitamin C/d as slow- or plain-release formulations together with 182 mg vitamin E/d for 4 weeks. The mean baseline levels of DNA repair incisions were 65.2 (95 % CI 60.4, 70.0) and 86.1 (95 % CI 76.2, 99.9) among the male smokers and well-nourished subjects, respectively. The male smokers also had high baseline levels of oxidised guanines in MNBC. After supplementation, only the male smokers supplemented with slow-release vitamin C tablets had increased DNA repair activity (27 (95 % CI 12, 41) % higher incision activity). These subjects also benefited from the supplementation by reduced levels of oxidised guanines in MNBC. In conclusion, nutritional status, DNA repair activity and DNA damage are linked, and beneficial effects of antioxidants might only be observed among poorly nourished subjects with high levels of oxidised DNA damage and low repair activity.

Effects of seven chemicals on DNA damage in the rat urinary bladder: a comet assay study.

PubMed

Wada, Kunio; Yoshida, Toshinori; Takahashi, Naofumi; Matsumoto, Kyomu

2014-07-15

The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained. Copyright © 2014 Elsevier B.V. All rights reserved.

The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells.

PubMed

Cho, Eun-Ah; Juhnn, Yong-Sung

2012-06-01

Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

[Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development].

PubMed

Bellé, Robert; Le Bouffant, Ronan; Morales, Julia; Cosson, Bertrand; Cormier, Patrick; Mulner-Lorillon, Odile

2007-01-01

Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy.

The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Eun-Ah; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

2012-06-01

Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNAmore » repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.« less

Oxidative damage and antioxidant defense in thymus of malnourished lactating rats.

PubMed

Gavia-García, Graciela; González-Martínez, Haydeé; Miliar-García, Ángel; Bonilla-González, Edmundo; Rosas-Trejo, María de Los Ángeles; Königsberg, Mina; Nájera-Medina, Oralia; Luna-López, Armando; González-Torres, María Cristina

2015-01-01

Malnutrition has been associated with oxidative damage by altered antioxidant protection mechanisms. Specifically, the aim of this study was to evaluate oxidative damage (DNA and lipid) and antioxidant status (superoxide dismutase [SOD], glutathione peroxidase [GPx], and catalase [CAT] mRNA, and protein expression) in thymus from malnourished rat pups. Malnutrition was induced during the lactation period by the food competition method. Oxidative DNA damage was determined quantifying 8-oxo-7, 8-dihydro-2'-deoxyguanosine adduct by high-performance liquid chromatography. Lipid peroxidation was assessed by the formation of thiobarbituric acid-reactive substances. Levels of gene and protein expression of SOD, GPx, and CAT were evaluated by real-time polymerase chain reaction and Western blot, respectively. Antioxidant enzyme activities were measured spectrophotometrically. Oxidative DNA damage and lipid peroxidation significantly increased in second-degree (MN-2) and third-degree malnourished (MN-3) rats compared with well-nourished rats. Higher amounts of oxidative damage, lower mRNA expression, and lower relative concentrations of protein, as well as decreased antioxidant activity of SOD, GPx, and CAT were associated with the MN-2 and MN-3 groups. The results of this study demonstrated that higher body-weight deficits were related to alterations in antioxidant protection, which contribute to increased levels of damage in the thymus. To our knowledge, this study demonstrated for the first time that early in life, malnutrition leads to increased DNA and lipid oxidative damage, attributable to damaged antioxidant mechanisms including transcriptional and enzymatic activity alterations. These findings may contribute to the elucidation of the causes of previously reported thymus dysfunction, and might explain partially why children and adults who have overcome child undernourishment experience immunologic deficiencies. Copyright © 2015 Elsevier Inc. All rights reserved.

Spatial Dependence of DNA Damage in Bacteria due to Low-Temperature Plasma Application as Assessed at the Single Cell Level

NASA Astrophysics Data System (ADS)

Privat-Maldonado, Angela; O'Connell, Deborah; Welch, Emma; Vann, Roddy; van der Woude, Marjan W.

2016-10-01

Low temperature plasmas (LTPs) generate a cocktail of reactive nitrogen and oxygen species (RNOS) with bactericidal activity. The RNOS however are spatially unevenly distributed in the plasma. Here we test the hypothesis that this distribution will affect the mechanisms underpinning plasma bactericidal activity focussing on the level of DNA damage in situ. For the first time, a quantitative, single cell approach was applied to assess the level of DNA damage in bacteria as a function of the radial distance from the centre of the plasma jet. Salmonella enterica on a solid, dry surface was treated with two types of LTP: an atmospheric-pressure dielectric barrier discharge plasma jet (charged and neutral species) and a radio-frequency atmospheric-pressure plasma jet (neutral species). In both cases, there was an inverse correlation between the degree of DNA damage and the radial distance from the centre of the plasma, with the highest DNA damage occurring directly under the plasma. This trend was also observed with Staphylococcus aureus. LTP-generated UV radiation was eliminated as a contributing factor. Thus valuable mechanistic information can be obtained from assays on biological material, which can inform the development of LTP as a complementary or alternative therapy for (topical) bacterial infections.

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.

Spatial Dependence of DNA Damage in Bacteria due to Low-Temperature Plasma Application as Assessed at the Single Cell Level

PubMed Central

Privat-Maldonado, Angela; O’Connell, Deborah; Welch, Emma; Vann, Roddy; van der Woude, Marjan W.

2016-01-01

Low temperature plasmas (LTPs) generate a cocktail of reactive nitrogen and oxygen species (RNOS) with bactericidal activity. The RNOS however are spatially unevenly distributed in the plasma. Here we test the hypothesis that this distribution will affect the mechanisms underpinning plasma bactericidal activity focussing on the level of DNA damage in situ. For the first time, a quantitative, single cell approach was applied to assess the level of DNA damage in bacteria as a function of the radial distance from the centre of the plasma jet. Salmonella enterica on a solid, dry surface was treated with two types of LTP: an atmospheric-pressure dielectric barrier discharge plasma jet (charged and neutral species) and a radio-frequency atmospheric-pressure plasma jet (neutral species). In both cases, there was an inverse correlation between the degree of DNA damage and the radial distance from the centre of the plasma, with the highest DNA damage occurring directly under the plasma. This trend was also observed with Staphylococcus aureus. LTP-generated UV radiation was eliminated as a contributing factor. Thus valuable mechanistic information can be obtained from assays on biological material, which can inform the development of LTP as a complementary or alternative therapy for (topical) bacterial infections. PMID:27759098

Rapid DNA double-strand breaks resulting from processing of Cr-DNA cross-links by both MutS dimers.

PubMed

Reynolds, Mindy F; Peterson-Roth, Elizabeth C; Bespalov, Ivan A; Johnston, Tatiana; Gurel, Volkan M; Menard, Haley L; Zhitkovich, Anatoly

2009-02-01

Mismatch repair (MMR) strongly enhances cyto- and genotoxicity of several chemotherapeutic agents and environmental carcinogens. DNA double-strand breaks (DSB) formed after two replication cycles play a major role in MMR-dependent cell death by DNA alkylating drugs. Here, we examined DNA damage detection and the mechanisms of the unusually rapid induction of DSB by MMR proteins in response to carcinogenic chromium(VI). We found that MSH2-MSH6 (MutSalpha) dimer effectively bound DNA probes containing ascorbate-Cr-DNA and cysteine-Cr-DNA cross-links. Binary Cr-DNA adducts, the most abundant form of Cr-DNA damage, were poor substrates for MSH2-MSH6, and their toxicity in cells was weak and MMR independent. Although not involved in the initial recognition of Cr-DNA damage, MSH2-MSH3 (MutSbeta) complex was essential for the induction of DSB, micronuclei, and apoptosis in human cells by chromate. In situ fractionation of Cr-treated cells revealed MSH6 and MSH3 chromatin foci that originated in late S phase and did not require replication of damaged DNA. Formation of MSH3 foci was MSH6 and MLH1 dependent, whereas MSH6 foci were unaffected by MSH3 status. DSB production was associated with progression of cells from S into G(2) phase and was completely blocked by the DNA synthesis inhibitor aphidicolin. Interestingly, chromosome 3 transfer into MSH3-null HCT116 cells activated an alternative, MSH3-like activity that restored dinucleotide repeat stability and sensitivity to chromate. Thus, sequential recruitment and unprecedented cooperation of MutSalpha and MutSbeta branches of MMR in processing of Cr-DNA cross-links is the main cause of DSB and chromosomal breakage at low and moderate Cr(VI) doses.

Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

NASA Astrophysics Data System (ADS)

Bazak, Remon; Ressl, Jan; Raha, Sumita; Doty, Caroline; Liu, William; Wanzer, Beau; Salam, Seddik Abdel; Elwany, Samy; Paunesku, Tatjana; Woloschak, Gayle E.

2013-11-01

A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells. Electronic supplementary information (ESI) available: http://janus.northwestern.edu/wololab/auxiliary/supplementary_data_2013.docx. See DOI: 10.1039/c3nr02203j

Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response.

PubMed

Cazzalini, Ornella; Scovassi, A Ivana; Savio, Monica; Stivala, Lucia A; Prosperi, Ennio

2010-01-01

Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21(CDKN1A) plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation. In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response. 2010 Elsevier B.V. All rights reserved.

ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress*

PubMed Central

Kemp, Michael G.; Sancar, Aziz

2016-01-01

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase that maintains genome stability and halts cell cycle phase transitions in response to DNA lesions that block DNA polymerase movement. These DNA replication-associated features of ATR function have led to the emergence of ATR kinase inhibitors as potential adjuvants for DNA-damaging cancer chemotherapeutics. However, whether ATR affects the genotoxic stress response in non-replicating, non-cycling cells is currently unknown. We therefore used chemical inhibition of ATR kinase activity to examine the role of ATR in quiescent human cells. Although ATR inhibition had no obvious effects on the viability of non-cycling cells, inhibition of ATR partially protected non-replicating cells from the lethal effects of UV and UV mimetics. Analyses of various DNA damage response signaling pathways demonstrated that ATR inhibition reduced the activation of apoptotic signaling by these agents in non-cycling cells. The pro-apoptosis/cell death function of ATR is likely due to transcription stress because the lethal effects of compounds that block RNA polymerase movement were reduced in the presence of an ATR inhibitor. These results therefore suggest that whereas DNA polymerase stalling at DNA lesions activates ATR to protect cell viability and prevent apoptosis, the stalling of RNA polymerases instead activates ATR to induce an apoptotic form of cell death in non-cycling cells. These results have important implications regarding the use of ATR inhibitors in cancer chemotherapy regimens. PMID:26940878

Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xuejiao; Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000; Zhang, Zhan

Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose-more » and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8-MOP.« less

DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

PubMed Central

Jagot-Lacoussiere, Léonard; Faye, Audrey; Bruzzoni-Giovanelli, Heriberto; Villoutreix, Bruno O; Rain, Jean-Christophe; Poyet, Jean-Luc

2015-01-01

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response. PMID:25695197

Significant accumulation of persistent organic pollutants and dysregulation in multiple DNA damage repair pathways in the electronic-waste-exposed populations

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Xiaobo; Jing, Yaqing; Wang, Jianhai

Electronic waste (e-waste) has created a worldwide environmental and health problem, by generating a diverse group of hazardous compounds such as persistent organic pollutants (POPs). Our previous studies demonstrated that populations from e-waste exposed region have a significantly higher level of chromosomal aberrancy and incidence of DNA damage. In this study, we further demonstrated that various POPs persisted at a significantly higher concentration in the exposed group than those in the unexposed group. The level of reactive oxygen species and micronucleus rate were also significantly elevated in the exposed group. RNA sequencing analysis revealed 31 genes in DNA damage responsesmore » and repair pathways that were differentially expressed between the two groups (Log 2 ratio >1 or <−1). Our data demonstrated that both females and males of the exposed group have activated a series of DNA damage response genes; however many important DNA repair pathways have been dysregulated. Expressions of NEIL1/3 and RPA3, which are critical in initiating base pair and nucleotide excision repairs respectively, have been downregulated in both females and males of the exposed group. In contrast, expression of RNF8, an E3 ligase involved in an error prone non-homologous end joining repair for DNA double strand break, was upregulated in both genders of the exposed group. The other genes appeared to be differentially expressed only when the males or females of the two groups were compared respectively. Importantly, the expression of cell cycle regulatory gene CDC25A that has been implicated in multiple kinds of malignant transformation was significantly upregulated among the exposed males while downregulated among the exposed females. In conclusion, our studies have demonstrated significant correlations between e-waste disposing and POPs accumulation, DNA lesions and dysregulation of multiple DNA damage repair mechanisms in the residents of the e-waste exposed region. - Highlights: • We compared concentration of POPs, ROS and micronucleus rate in POPs exposed area. • Significant accumulation of POPs homologous in the e-waste exposed residents. • DNA damage and DNA damage repair pathways have been differentially activated. • Females and males in the exposed group have different responses to the DNA damage. • Exposed males may be more prone to undergo malignant transformation.« less

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

PubMed Central

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

DNA Damage Protecting Activity and Antioxidant Potential of Launaea taraxacifolia Leaves Extract.

PubMed

Adinortey, Michael Buenor; Ansah, Charles; Weremfo, Alexander; Adinortey, Cynthia Ayefoumi; Adukpo, Genevieve Etornam; Ameyaw, Elvis Ofori; Nyarko, Alexander Kwadwo

2018-01-01

The leaf extract of Launaea taraxacifolia commonly known as African Lettuce is used locally to treat dyslipidemia and liver diseases, which are associated with oxidative stress. Methanol extract from L. taraxacifolia leaves was tested for its antioxidant activity and its ability to protect DNA from oxidative damage. In vitro antioxidant potential of the leaf extract was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), and hydroxyl (OH) radical scavenging assays. Ferric reducing power, total antioxidant capacity (TAC), metal chelating, and anti-lipid peroxidation ability of the extract were also examined using gallic acid, ascorbic acid, citric acid, and ethylenediaminetetraacetic acid as standards. L. taraxacifolia leaves extract showed antioxidant activity with IC 50 values of 16.18 μg/ml (DPPH), 123.3 μg/ml (NO), 128.2 μg/ml (OH radical), 97.94 μg/ml (metal chelating), 80.28 μg/ml (TAC), and 23 μg/ml (anti-lipid peroxidation activity). L. taraxacifolia leaves extract exhibited a strong capability for DNA damage protection at 20 mg/ml concentration. These findings suggest that the methanolic leaf extract of L. taraxacifolia could be used as a natural antioxidant and also as a preventive therapy against diseases such as arteriosclerosis associated with DNA damage.

SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes.

PubMed

Calapre, Leslie; Gray, Elin S; Kurdykowski, Sandrine; David, Anthony; Descargues, Pascal; Ziman, Mel

2017-06-10

Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes. Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin. This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jarvis, Ian W.H., E-mail: Ian.Jarvis@ki.se; Bergvall, Christoffer, E-mail: Christoffer.Bergvall@anchem.su.se; Bottai, Matteo, E-mail: Matteo.Bottai@ki.se

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 DNAmore » 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.« less

New orally active DNA minor groove binding small molecule CT-1 acts against breast cancer by targeting tumor DNA damage leading to p53-dependent apoptosis.

PubMed

Saini, Karan Singh; Hamidullah; Ashraf, Raghib; Mandalapu, Dhanaraju; Das, Sharmistha; Siddiqui, Mohd Quadir; Dwivedi, Sonam; Sarkar, Jayanta; Sharma, Vishnu Lal; Konwar, Rituraj

2017-04-01

Targeting tumor DNA damage and p53 pathway is a clinically established strategy in the development of cancer chemotherapeutics. Majority of anti-cancer drugs are delivered through parenteral route for reasons like severe toxicity, lack of stability, and poor enteral absorption. Current DNA targeting drugs in clinical like anthracycline suffers from major drawbacks like cardiotoxicity. Here, we report identification of a new orally active small molecule curcumin-triazole conjugate (CT-1) with significant anti-breast cancer activity in vitro and in vivo. CT-1 selectively and significantly inhibits viability of breast cancer cell lines; retards cells cycle progression at S phase and induce mitochondrial-mediated cell apoptosis. CT-1 selectively binds to minor groove of DNA and induces DNA damage leading to increase in p53 along with decrease in its ubiquitination. Inhibition of p53 with pharmacological inhibitor as well as siRNA revealed the necessity of p53 in CT-1-mediated anti-cancer effects in breast cancer cells. Studies using several other intact p53 and deficient p53 cancer cell lines further confirmed necessity of p53 in CT-1-mediated anti-cancer response. Pharmacological inhibition of pan-caspase showed CT-1 induces caspase-dependent cell death in breast cancer cells. Most interestingly, oral administration of CT-1 induces significant inhibition of tumor growth in LA-7 syngeneic orthotropic rat mammary tumor model. CT-1 treated mammary tumor shows enhancement in DNA damage, p53 upregulation, and apoptosis. Collectively, CT-1 exhibits potent anti-cancer effect both in vitro and in vivo and could serve as a safe orally active lead for anti-cancer drug development. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Interaction between Checkpoint Kinase 1 (Chk1) and the Minichromosome Maintenance (MCM) Complex Is Required for DNA Damage-induced Chk1 Phosphorylation*

PubMed Central

Han, Xiangzi; Aslanian, Aaron; Fu, Kang; Tsuji, Toshiya; Zhang, Youwei

2014-01-01

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage. PMID:25049228

The p53–Mdm2 feedback loop protects against DNA damage by inhibiting p53 activity but is dispensable for p53 stability, development, and longevity

PubMed Central

Pant, Vinod; Xiong, Shunbin; Jackson, James G.; Post, Sean M.; Abbas, Hussein A.; Quintás-Cardama, Alfonso; Hamir, Amirali N.; Lozano, Guillermina

2013-01-01

The p53–Mdm2 feedback loop is perceived to be critical for regulating stress-induced p53 activity and levels. However, this has never been tested in vivo. Using a genetically engineered mouse with mutated p53 response elements in the Mdm2 P2 promoter, we show that feedback loop-deficient Mdm2P2/P2 mice are viable and aphenotypic and age normally. p53 degradation kinetics after DNA damage in radiosensitive tissues remains similar to wild-type controls. Nonetheless, DNA damage response is elevated in Mdm2P2/P2 mice. Enhanced p53-dependent apoptosis sensitizes hematopoietic stem cells (HSCs), causing drastic myeloablation and lethality. These results suggest that while basal Mdm2 levels are sufficient to regulate p53 in most tissues under homeostatic conditions, the p53–Mdm2 feedback loop is critical for regulating p53 activity and sustaining HSC function after DNA damage. Therefore, transient disruption of p53–Mdm2 interaction could be explored as a potential adjuvant/therapeutic strategy for targeting stem cells in hematological malignancies. PMID:23973961

The Intertwined Roles of Transcription and Repair Proteins

PubMed Central

Fong, Yick W.; Cattoglio, Claudia; Tjian, Robert

2014-01-01

Transcription is apparently risky business. Its intrinsic mutagenic potential must be kept in check by networks of DNA repair factors that monitor the transcription process to repair DNA lesions that could otherwise compromise transcriptional fidelity and genome integrity. Intriguingly, recent studies point to an even more direct function of DNA repair complexes as co-activators of transcription and the unexpected role of “scheduled” DNA damage/repair at gene promoters. Paradoxically, spontaneous DNA double-strand breaks also induce ectopic transcription that is essential for repair. Thus, transcription, DNA damage and repair may be more physically and functionally intertwined than previously appreciated. PMID:24207023

Exposure to Ultrafine Particles from Ambient Air and Oxidative Stress–Induced DNA Damage

PubMed Central

Bräuner, Elvira Vaclavik; Forchhammer, Lykke; Møller, Peter; Simonsen, Jacob; Glasius, Marianne; Wåhlin, Peter; Raaschou-Nielsen, Ole; Loft, Steffen

2007-01-01

Background Particulate matter, especially ultrafine particles (UFPs), may cause health effects through generation of oxidative stress, with resulting damage to DNA and other macromolecules. Objective We investigated oxidative damage to DNA and related repair capacity in peripheral blood mononuclear cells (PBMCs) during controlled exposure to urban air particles with assignment of number concentration (NC) to four size modes with average diameters of 12, 23, 57, and 212 nm. Design Twenty-nine healthy adults participated in a randomized, two-factor cross-over study with or without biking exercise for 180 min and with exposure to particles (NC 6169-15362/cm3) or filtered air (NC 91-542/cm3) for 24 hr. Methods The levels of DNA strand breaks (SBs), oxidized purines as formamidopyrimidine DNA glycolase (FPG) sites, and activity of 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1) in PBMCs were measured by the Comet assay. mRNA levels of OGG1, nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), and heme oxygenase-1 (HO1) were determined by real-time reverse transcriptase–polymerase chain reaction. Results Exposure to UFPs for 6 and 24 hr significantly increased the levels of SBs and FPG sites, with a further insignificant increase after physical exercise. The OGG1 activity and expression of OGG1, NUDT1, and HO1 were unaltered. There was a significant dose–response relationship between NC and DNA damage, with the 57-nm mode as the major contributor to effects. Concomitant exposure to ozone, nitrogen oxides, and carbon monoxide had no influence. Conclusion Our results indicate that UFPs, especially the 57-nm soot fraction from vehicle emissions, causes systemic oxidative stress with damage to DNA and no apparent compensatory up-regulation of DNA repair within 24 hr. PMID:17687444

Mitochondria damage checkpoint in apoptosis and genome stability.

PubMed

Singh, Keshav K

2004-11-01

Mitochondria perform multiple cellular functions including energy production, cell proliferation and apoptosis. Studies described in this paper suggest a role for mitochondria in maintaining genomic stability. Genomic stability appears to be dependent on mitochondrial functions involved in maintenance of proper intracellular redox status, ATP-dependent transcription, DNA replication, DNA repair and DNA recombination. To further elucidate the role of mitochondria in genomic stability, I propose a mitochondria damage checkpoint (mitocheckpoint) that monitors and responds to damaged mitochondria. Mitocheckpoint can coordinate and maintain proper balance between apoptotic and anti-apoptotic signals. When mitochondria are damaged, mitocheckpoint can be activated to help cells repair damaged mitochondria, to restore normal mitochondrial function and avoid production of mitochondria-defective cells. If mitochondria are severely damaged, mitocheckpoint may not be able to repair the damage and protect cells. Such an event triggers apoptosis. If damage to mitochondria is continuous or persistent such as damage to mitochondrial DNA resulting in mutations, mitocheckpoint may fail which can lead to genomic instability and increased cell survival in yeast. In human it can cause cancer. In support of this proposal we provide evidence that mitochondrial genetic defects in both yeast and mammalian systems lead to impaired DNA repair, increased genomic instability and increased cell survival. This study reveals molecular genetic mechanisms underlying a role for mitochondria in carcinogenesis in humans.

Novel neuroprotective and hepatoprotective effects of citric acid in acute malathion intoxication.

PubMed

Abdel-Salam, Omar M E; Youness, Eman R; Mohammed, Nadia A; Yassen, Noha N; Khadrawy, Yasser A; El-Toukhy, Safinaz Ebrahim; Sleem, Amany A

2016-12-01

To study the effect of citric acid given alone or combined with atropine on brain oxidative stress, neuronal injury, liver damage, and DNA damage of peripheral blood lymphocytes induced in the rat by acute malathion exposure. Rats were received intraperitoneal (i.p.) injection of malathion 150 mg/kg along with citric acid (200 or 400 mg/kg, orally), atropine (1 mg/kg, i.p.) or citric acid 200 mg/kg + atropine 1 mg/kg and euthanized 4 h later. Malathion resulted in increased lipid peroxidation (malondialdehyde) and nitric oxide concentrations accompanied with a decrease in brain reduced glutathione, glutathione peroxidase (GPx) activity, total antioxidant capacity (TAC) and glucose concentrations. Paraoxonase-1, acetylcholinesterase (AChE) and butyrylcholinesterase activities decreased in brain as well. Liver aspartate aminotransferase and alanine aminotransferase activities were raised. The comet assay showed increased DNA damage of peripheral blood lymphocytes. Histological damage and increased expression of inducible nitric oxide synthase (iNOS) were observed in brain and liver. Citric acid resulted in decreased brain lipid peroxidation and nitric oxide. Meanwhile, glutathione, GPx activity, TAC capacity and brain glucose level increased. Brain AChE increased but PON1 and butyrylcholinesterase activities decreased by citric acid. Liver enzymes, the percentage of damaged blood lymphocytes, histopathological alterations and iNOS expression in brain and liver was decreased by citric acid. Meanwhile, rats treated with atropine showed decreased brain MDA, nitrite but increased GPx activity, TAC, AChE and glucose. The drug also decreased DNA damage of peripheral blood lymphocytes, histopathological alterations and iNOS expression in brain and liver. The study demonstrates a beneficial effect for citric acid upon brain oxidative stress, neuronal injury, liver and DNA damage due to acute malathion exposure. Copyright © 2016 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors*

PubMed Central

Tapias, Angels; Auriol, Jerome; Forget, Diane; Enzlin, Jacqueline H.; Schärer, Orlando D; Coin, Frederic; Coulombe, Benoit; Egly, Jean-Marc

2015-01-01

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position −3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position −6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position −19 to +8. XPG and XPF/ERCC1 endo-nucleases then cut the damaged DNA at the limit of the opened structure that was previously “labeled” by the positioning of XPC/HR23B and TFIIH. PMID:14981083

Sulforaphane inhibits CYP1A1 activity and promotes genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Fangxing, E-mail: fxyang@zju.edu.cn; Zhuang, Shulin; Zhang, Chao

2013-06-15

Increasing environmental pollution by carcinogens such as some of persistent organic pollutants (POPs) has prompted growing interest in searching for chemopreventive compounds which are readily obtainable. Sulforaphane (SFN) is isolated from cruciferous vegetables and has the potentials to reduce carcinogenesis through various pathways. In this study, we studied the effects of SFN on CYP1A1 activity and genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that SFN inhibited TCDD-induced CYP1A1 activity in H4IIE cells by directly inhibiting CYP1A1 activity, probably through binding to aryl hydrocarbon receptor and/or CYP1A1 revealed by molecular docking. However, SFN promoted TCDD-induced DNA damage in yeast cellsmore » and reduced the viability of initiated yeast cells. Besides, it is surprising that SFN also failed to reduce genotoxicity induced by other genotoxic reagents which possess different mechanisms to lead to DNA damage. Currently, it is difficult to predict whether SFN has the potentials to reduce the risk of TCDD based on the conflicting observations in the study. Therefore, further studies should be urgent to reveal the function and mechanism of SFN in the stress of such POPs on human health. - Highlights: • Sulforaphane inhibited TCDD-induced CYP1A1 activity in H4IIE cells. • Sulforaphane may bind to aryl hydrocarbon receptor and/or CYP1A1. • Sulforaphane promoted TCDD-induced DNA damage in yeast cells. • Sulforaphane may promote DNA damage by DNA strand breaks or DNA alkylation.« less

Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Satyender; Kumar, Vivek; Vashisht, Kapil

2011-11-15

Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activitymore » toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 {+-} 2.15 vs. 6.24 {+-} 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. -- Highlights: Black-Right-Pointing-Pointer Role of CYP1A1, CYP3A5, CYP2C, CYP2D6 and PON1 genotypes on DNA damage. Black-Right-Pointing-Pointer Workers exposed to some OPs demonstrated increased DNA damage. Black-Right-Pointing-Pointer CYP2D6 *3 PM and PON1 (Q192R and L55M) genotypes are associated with DNA damage. Black-Right-Pointing-Pointer Concomitant presence of certain CYP2D6 and PON1 genotypes can increase DNA damage.« less

Caspase 3 promotes genetic instability and carcinogenesis

PubMed Central

Liu, Xinjian; He, Yujun; Li, Fang; Huang, Qian; Kato, Takamitsu A.; Hall, Russell P; Li, Chuan-Yuan

2015-01-01

Summary Apoptosis is typically considered an anti-oncogenic process since caspase activation can promote the elimination of genetically unstable or damaged cells. We report that a central effector of apoptosis, caspase 3, facilitates, rather than suppresses, chemical and radiation-induced genetic instability and carcinogenesis. We found that a significant fraction of mammalian cells treated with ionizing radiation can survive, despite caspase 3 activation. Moreover, this sublethal activation of caspase 3 promoted persistent DNA damage and oncogenic transformation. In addition, chemically-induced skin carcinogenesis was significantly reduced in mice genetically deficient in caspase 3. Furthermore, attenuation of Endo G activity significantly reduced radiation-induced DNA damage and oncogenic transformation, identifying Endo G as a downstream effector of caspase 3 in this pathway. Our findings suggest that rather than acting as a broad inhibitor of carcinogenesis, caspase 3 activation may contribute to genome instability and play a pivotal role in tumor formation following damage. PMID:25866249

Oxidative damage and cell-programmed death induced in Zea mays L. by allelochemical stress.

PubMed

Ciniglia, Claudia; Mastrobuoni, Francesco; Scortichini, Marco; Petriccione, Milena

2015-05-01

The allelochemical stress on Zea mays was analyzed by using walnut husk washing waters (WHWW), a by-product of Juglans regia post-harvest process, which possesses strong allelopathic potential and phytotoxic effects. Oxidative damage and cell-programmed death were induced by WHWW in roots of maize seedlings. Treatment induced ROS burst, with excess of H2O2 content. Enzymatic activities of catalase were strongly increased during the first hours of exposure. The excess in malonildialdehyde following exposure to WHWW confirmed that oxidative stress severely damaged maize roots. Membrane alteration caused a decrease in NADPH oxidase activity along with DNA damage as confirmed by DNA laddering. The DNA instability was also assessed through sequence-related amplified polymorphism assay, thus suggesting the danger of walnut processing by-product and focusing the attention on the necessity of an efficient treatment of WHWW.

What’s the Damage? The Impact of Pathogens on Pathways that Maintain Host Genome Integrity

PubMed Central

Weitzman, Matthew D.; Weitzman, Jonathan B.

2014-01-01

Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs. PMID:24629335

Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo. [UV radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanaka, K.; Hayakawa, H.; Sekiguchi, M.

1977-07-01

The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by uv light. A clear dose response was observed between the level of uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4v/sub 1/, a mutant defective in the endonuclease V gene, showed no ability to restore themore » uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by uv light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after uv irradiation.« less

Co-expression of antioxidant enzymes with expression of p53, DNA repair, and heat shock protein genes in the gamma ray-irradiated hermaphroditic fish Kryptolebias marmoratus larvae.

PubMed

Rhee, Jae-Sung; Kim, Bo-Mi; Kim, Ryeo-Ok; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

2013-09-15

To investigate effects of gamma ray irradiation in the hermaphroditic fish, Kryptolebias marmoratus larvae, we checked expression of p53, DNA repair, and heat shock protein genes with several antioxidant enzyme activities by quantitative real-time RT-PCR and biochemical methods in response to different doses of gamma radiation. As a result, the level of gamma radiation-induced DNA damage was initiated after 4Gy of radiation, and biochemical and molecular damage became substantial from 8Gy. In particular, several DNA repair mechanism-related genes were significantly modulated in the 6Gy gamma radiation-exposed fish larvae, suggesting that upregulation of such DNA repair genes was closely associated with cell survival after gamma irradiation. The mRNA expression of p53 and most hsps was also significantly upregulated at high doses of gamma radiation related to cellular damage. This finding indicates that gamma radiation can induce oxidative stress with associated antioxidant enzyme activities, and linked to modulation of the expression of DNA repair-related genes as one of the defense mechanisms against radiation damage. This study provides a better understanding of the molecular mode of action of defense mechanisms upon gamma radiation in fish larvae. Copyright © 2013 Elsevier B.V. All rights reserved.

Roles of nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage.

PubMed

Marcelain, Katherine; De La Torre, Consuelo; González, Patricio; Pincheira, Juana

2005-01-01

Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.

ICAM-1 and AMPK regulate cell detachment and apoptosis by N-methyl-N Prime -nitro-N-nitrosoguanidine, a widely spread environmental chemical, in human hormone-refractory prostate cancers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yi-Cheng; Lu, Pin-Hsuan; Hsu, Jui-Ling

2011-12-15

Poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, plays a crucial role in the regulation of DNA repair. PARP-1 hyperactivation causes DNA damage and cell death. The underlying mechanism is complicated and is through diverse pathways. The understanding of responsible signaling pathways may offer implications for effective therapies. After concentration-response determination of N-Methyl-N Prime -Nitro-N-Nitrosoguanidine (MNNG, a PARP-1 activating agent and an environmental mutagen) in human hormone-refractory prostate cancers, the data showed that concentrations below 5 {mu}M did not change cell survival but cause a time-dependent up-regulation of intracellular adhesion molecule-1 (ICAM-1) in mRNA, total protein and cell surface levels.more » Detection of phosphorylation and degradation of I{kappa}B-{alpha} and nuclear translocation of NF-{kappa}B showed that MNNG induced the activation of NF-{kappa}B that was responsible for the ICAM-1 up-regulation since PDTC (a NF-{kappa}B inhibitor) significantly abolished this effect. However, higher concentrations (e.g., 10 {mu}M) of MNNG induced a 61% detachment of the cells which were apoptosis associated with the activation of AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Further identification showed that both AMPK and JNK other than p38 MAPK functionally contributed to cell death. The remaining 39% attached cells were survival associated with high ICAM-1 expression. In conclusion, the data suggest that NF-{kappa}B-dependent up-regulation of ICAM-1 plays a key role on cell attachment and survival; whereas, activation of AMPK and JNK participates in cytotoxic signaling pathways in detached cells caused by PARP-1 activation. Highlights: Black-Right-Pointing-Pointer Low level of DNA damage helps cell attachment and survival via ICAM-1 upregulation. Black-Right-Pointing-Pointer High level of DNA damage causes AMPK- and JNK-involved cell detachment and death. Black-Right-Pointing-Pointer The study provides an anticancer approach targeting PARP-1 and DNA damage response.« less

DNA damage in lead-exposed hepatocytes: coexistence of apoptosis and necrosis?

PubMed

Narayana, Kilarkaje; Raghupathy, Raj

2012-04-01

The aim of the present study was to investigate the coexistence of oxidative DNA damage and apoptosis- and necrosis-related DNA damage, and to correlate with ultrastructural changes in hepatocyte nuclei in the lead-nitrate-exposed liver. Adult male Wistar rats were exposed to 0, 0.5, and 1% lead nitrate for 60 days, and the livers were sampled the next day. Ultrastructurally, hepatocyte nuclei showed no apoptosis-related morphological changes, but showed necrotic changes. Competitive enzyme-linked immunosorbent assay showed no change in 8-oxo-dG activity (P > 0.05), but immunohistochemistry showed its localization in hepatocytes, Kupffer cells, endothelium, and bile ductule epithelium. TUNEL-labeled DNA breaks presenting 3'-OH ends increased in hepatocytes in all functional zones of the portal acini and bile ductule epithelium (zones I>III>II). In situ oligo ligation revealed the existence of DNA breaks bearing duplex 3' overhangs and 5' P-blunt ends in hepatocytes of all functional zones and bile ductule epithelium. In conclusion, both apoptosis- and necrosis-related DNA damage coexist without significant oxidative DNA damage. Hepatocytes display changes related to necrosis, but not those related to apoptosis.

SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

PubMed Central

Kim, Seung-Hwan; Holway, Antonia H.; Wolff, Suzanne; Dillin, Andrew; Michael, W. Matthew

2007-01-01

During early embryogenesis in Caenorhabditis elegans, the ATL-1–CHK-1 (ataxia telangiectasia mutated and Rad3 related–Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1–PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context. PMID:17908915

Chk2 and REGγ-dependent DBC1 regulation in DNA damage induced apoptosis

PubMed Central

Magni, Martina; Ruscica, Vincenzo; Buscemi, Giacomo; Kim, Ja-Eun; Nachimuthu, Benjamin Tamilselvan; Fontanella, Enrico; Delia, Domenico; Zannini, Laura

2014-01-01

Human DBC1 (Deleted in Breast Cancer 1; KIAA1967; CCAR2) is a protein implicated in the regulation of apoptosis, transcription and histone modifications. Upon DNA damage, DBC1 is phosphorylated by ATM/ATR on Thr454 and this modification increases its inhibitory interaction with SIRT1, leading to p53 acetylation and p53-dependent apoptosis. Here, we report that the inhibition of SIRT1 by DBC1 in the DNA damage response (DDR) also depends on Chk2, the transducer kinase that is activated by ATM upon DNA lesions and contributes to the spreading of DNA damage signal. Indeed we found that inactivation of Chk2 reduces DBC1-SIRT1 binding, thus preventing p53 acetylation and DBC1-induced apoptosis. These events are mediated by Chk2 phosphorylation of the 11S proteasome activator REGγ on Ser247, which increases REGγ-DBC1 interaction and SIRT1 inhibition. Overall our results clarify the mechanisms underlying the DBC1-dependent SIRT1 inhibition and link, for the first time, Chk2 and REGγ to the ATM-DBC1-SIRT1 axis. PMID:25361978

Bimodal regulation of p21waf1 protein as function of DNA damage levels

PubMed Central

Buscemi, G; Ricci, C; Zannini, L; Fontanella, E; Plevani, P; Delia, D

2014-01-01

Human p21Waf1 protein is well known for being transcriptionally induced by p53 and activating the cell cycle checkpoint arrest in response to DNA breaks. Here we report that p21Waf1 protein undergoes a bimodal regulation, being upregulated in response to low doses of DNA damage but rapidly and transiently degraded in response to high doses of DNA lesions. Responsible for this degradation is the checkpoint kinase Chk1, which phosphorylates p21Waf1 on T145 and S146 residues and induces its proteasome-dependent proteolysis. The initial p21Waf1 degradation is then counteracted by the ATM-Chk2 pathway, which promotes the p53-dependent accumulation of p21Waf1 at any dose of damage. We also found that p21Waf1 ablation favors the activation of an apoptotic program to eliminate otherwise irreparable cells. These findings support a model in which in human cells a balance between ATM-Chk2-p53 and the ATR-Chk1 pathways modulates p21Waf1 protein levels in relation to cytostatic and cytotoxic doses of DNA damage. PMID:25486478

Ultraviolet-B-induced DNA damage and ultraviolet-B tolerance mechanisms in species with different functional groups coexisting in subalpine moorlands.

PubMed

Wang, Qing-Wei; Kamiyama, Chiho; Hidema, Jun; Hikosaka, Kouki

2016-08-01

High doses of ultraviolet-B (UV-B; 280-315 nm) radiation can have detrimental effects on plants, and especially damage their DNA. Plants have DNA repair and protection mechanisms to prevent UV-B damage. However, it remains unclear how DNA damage and tolerance mechanisms vary among field species. We studied DNA damage and tolerance mechanisms in 26 species with different functional groups coexisting in two moorlands at two elevations. We collected current-year leaves in July and August, and determined accumulation of cyclobutane pyrimidine dimer (CPD) as UV-B damage and photorepair activity (PRA) and concentrations of UV-absorbing compounds (UACs) and carotenoids (CARs) as UV-B tolerance mechanisms. DNA damage was greater in dicot than in monocot species, and higher in herbaceous than in woody species. Evergreen species accumulated more CPDs than deciduous species. PRA was higher in Poaceae than in species of other families. UACs were significantly higher in woody than in herbaceous species. The CPD level was not explained by the mechanisms across species, but was significantly related to PRA and UACs when we ignored species with low CPD, PRA and UACs, implying the presence of another effective tolerance mechanism. UACs were correlated negatively with PRA and positively with CARs. Our results revealed that UV-induced DNA damage significantly varies among native species, and this variation is related to functional groups. DNA repair, rather than UV-B protection, dominates in UV-B tolerance in the field. Our findings also suggest that UV-B tolerance mechanisms vary among species under evolutionary trade-off and synergism.

The Replication Stress Response in Pancreatic Cancer

DTIC Science & Technology

2013-10-01

network that recognizes challenges to DNA replication and mobilizes diverse activities to maintain genome integrity. The RSR is critical for the...pancreatic cancer cells. We further validated positive hits be deconvolution of individual siRNAs and began work on determining their activities in DNA replication and DNA damage responses.

Inhibition of the Mitotic Exit Network in Response to Damaged Telomeres

PubMed Central

Valerio-Santiago, Mauricio; de los Santos-Velázquez, Ana Isabel; Monje-Casas, Fernando

2013-01-01

When chromosomal DNA is damaged, progression through the cell cycle is halted to provide the cells with time to repair the genetic material before it is distributed between the mother and daughter cells. In Saccharomyces cerevisiae, this cell cycle arrest occurs at the G2/M transition. However, it is also necessary to restrain exit from mitosis by maintaining Bfa1-Bub2, the inhibitor of the Mitotic Exit Network (MEN), in an active state. While the role of Bfa1 and Bub2 in the inhibition of mitotic exit when the spindle is not properly aligned and the spindle position checkpoint is activated has been extensively studied, the mechanism by which these proteins prevent MEN function after DNA damage is still unclear. Here, we propose that the inhibition of the MEN is specifically required when telomeres are damaged but it is not necessary to face all types of chromosomal DNA damage, which is in agreement with previous data in mammals suggesting the existence of a putative telomere-specific DNA damage response that inhibits mitotic exit. Furthermore, we demonstrate that the mechanism of MEN inhibition when telomeres are damaged relies on the Rad53-dependent inhibition of Bfa1 phosphorylation by the Polo-like kinase Cdc5, establishing a new key role of this kinase in regulating cell cycle progression. PMID:24130507

Activation of EGFR and ERBB2 by Helicobacter pylori Results in Survival of Gastric Epithelial Cells with DNA Damage

PubMed Central

Chaturvedi, Rupesh; Asim, Mohammad; Piazuelo, M. Blanca; Yan, Fang; Barry, Daniel P.; Sierra, Johanna Carolina; Delgado, Alberto G.; Hill, Salisha; Casero, Robert A.; Bravo, Luis E.; Dominguez, Ricardo L.; Correa, Pelayo; Polk, D. Brent; Washington, M. Kay; Rose, Kristie L.; Schey, Kevin L.; Morgan, Douglas R.; Peek, Richard M.; Wilson, Keith T.

2014-01-01

BACKGROUND & AIMS The gastric cancer-causing pathogen Helicobacter pylori upregulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOXhigh cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H pylori-infected Egfrwa5 mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. Phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsies from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS SMOX expression and DNA damage were decreased, and apoptosis increased in H pylori-infected Egfrwa5 mice. H pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damagehigh apoptosislow cells. Phosphoproteomic analysis revealed increased EGFR and ERBB2 signaling. Immunoblot analysis demonstrated the presence of a phosphorylated (p)EGFR–ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damagehigh apoptosislow cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR–ERBB2, and pERBB2 were increased predominantly in tissues demonstrating gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR–ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis. PMID:24530706

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawiak, Anna; Piosik, Jacek; Stasilojc, Grzegorz

2007-09-15

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topomore » II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.« less

Induction of a G1-S checkpoint in fission yeast.

PubMed

Bøe, Cathrine A; Krohn, Marit; Rødland, Gro Elise; Capiaghi, Christoph; Maillard, Olivier; Thoma, Fritz; Boye, Erik; Grallert, Beáta

2012-06-19

Entry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G(1)-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G(1)-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G(1)-S delay depends on at least two different inputs.

Mitochondrial DNA Damage Initiates Acute Lung Injury and Multi-Organ System Failure Evoked in Rats by Intra-Tracheal Pseudomonas Aeruginosa.

PubMed

Lee, Yann-Leei; Obiako, Boniface; Gorodnya, Olena M; Ruchko, Mykhaylo V; Kuck, Jamie L; Pastukh, Viktor M; Wilson, Glenn L; Simmons, Jon D; Gillespie, Mark N

2017-07-01

Although studies in rat cultured pulmonary artery endothelial cells, perfused lungs, and intact mice support the concept that oxidative mitochondrial (mt) DNA damage triggers acute lung injury (ALI), it has not yet been determined whether enhanced mtDNA repair forestalls development of ALI and its progression to multiple organ system failure (MOSF). Accordingly, here we examined the effect of a fusion protein construct targeting the DNA glycosylase, Ogg1, to mitochondria in a rat model intra-tracheal Pseudomonas aeruginosa (strain 103; PA103)-induced ALI and MOSF. Relative to controls, animals given PA103 displayed increases in lung vascular filtration coefficient accompanied by transient lung tissue oxidative mtDNA damage and variable changes in mtDNA copy number without evidence of nuclear DNA damage. The approximate 40% of animals surviving 24 h after bacterial administration exhibited multiple organ dysfunction, manifest as increased serum and tissue-specific indices of kidney and liver failure, along with depressed heart rate and blood pressure. While administration of mt-targeted Ogg1 to control animals was innocuous, the active fusion protein, but not a DNA repair-deficient mutant, prevented bacteria-induced increases in lung tissue oxidative mtDNA damage, failed to alter mtDNA copy number, and attenuated lung endothelial barrier degradation. These changes were associated with suppression of liver, kidney, and cardiovascular dysfunction and with decreased 24 h mortality. Collectively, the present findings indicate that oxidative mtDNA damage to lung tissue initiates PA103-induced ALI and MOSF in rats.

Adenovirus-mediated FIR demonstrated TP53-independent cell-killing effect and enhanced antitumor activity of carbon-ion beams.

PubMed

Kano, M; Matsushita, K; Rahmutulla, B; Yamada, S; Shimada, H; Kubo, S; Hiwasa, T; Matsubara, H; Nomura, F

2016-01-01

Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or γ rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by γH2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIRΔexon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P=0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late-phase severe adverse effects independently of the TP53 status in vitro. Our findings indicated the feasibility of the combination of Ad-FIR with DNA damaging agents for future esophageal cancer treatment.

Changes in pyruvate metabolism detected by magnetic resonance imaging are linked to DNA damage and serve as a sensor of temozolomide response in glioblastoma cells

PubMed Central

Park, Ilwoo; Mukherjee, Joydeep; Ito, Motokazu; Chaumeil, Myriam M.; Jalbert, Llewellyn E.; Gaensler, Karin; Ronen, Sabrina M.; Nelson, Sarah J.; Pieper, Russell O.

2014-01-01

Recent findings show that exposure to temozolomide (TMZ), a DNA damaging drug used to treat glioblastoma, can suppress the conversion of pyruvate to lactate. To understand the mechanistic basis for this effect and its potential utility as a TMZ response biomarker, we compared the response of isogenic glioblastoma cell populations differing only in expression of the DNA repair protein MGMT, a TMZ-sensitivity determinant, after exposure to TMZ in vitro and in vivo. Hyperpolarized [1-(13)C]-pyruvate-based magnetic resonance imaging was used to monitor temporal effects on pyruvate metabolism in parallel with DNA damage responses and tumor cell growth. TMZ exposure decreased conversion of pyruvate to lactate only in MGMT-deficient cells. This effect coincided temporally with TMZ-induced increases in levels of the DNA damage response protein pChk1. Changes in pyruvate to lactate conversion triggered by TMZ preceded tumor growth suppression and were not associated with changes in levels of NADH or lactate dehydrogenase activity in tumors. Instead, they were associated with a TMZ-induced decrease in the expression and activity of pyruvate kinase PKM2, a glycolytic enzyme that indirectly controls pyruvate metabolism. PKM2 silencing decreased pyruvate kinase activity, intracellular lactate levels, and conversion of pyruvate to lactate in the same manner as TMZ, and Chk1 silencing blocked the TMZ-induced decrease in PKM2 expression. Overall, our findings showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by magnetic resonance imaging methods as an early sensor of TMZ therapeutic response. PMID:25320009

The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

PubMed

Mathew, Shomita S; Bridge, Eileen

2007-09-01

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

NOD2 expression, DNA damage and oxido-inflammatory status in atopic bronchial asthma: Exploring their nexus to disease severity.

PubMed

Gaballah, Hanaa H; Gaber, Rasha A; Sharshar, Ragia S; Elshweikh, Samah A

2018-06-20

Allergic asthma is a chronically relapsing inflammatory airway disease with a complex pathophysiology. This study was undertaken to investigate the potential contribution of NOD2 signaling, proinflammatory cytokines, chitotriosidase (CHIT1) activity, oxidative stress and DNA damage to atopic asthma pathogenesis, as well as to explore their possible role as surrogate noninvasive biomarkers for monitoring asthma severity. Sixty patients with atopic bronchial asthma who were divided according to asthma severity into 40 mild-moderate, 20 severe atopic asthmatics, in addition to thirty age-matched healthy controls were enrolled in this study. NOD2 expression in PBMCs was assessed by quantitative real-time RT-PCR. DNA damage indices were assessed by alkaline comet assay. Serum IgE, IL-17, IL-8 and 3-Nitrotyrosine levels were estimated by ELISA. Serum CHIT1and GST activities, as well as MDA levels, were measured. NOD2 mRNA relative expression levels were significantly decreased in atopic asthmatic cases relative to controls with lower values among severe atopic asthmatics. On the other hand, IL-17 and IL-8 serum levels, CHIT1 activity, DNA damage indices and oxidative stress markers were significantly increased in atopic asthmatic cases relative to controls with higher values among severe atopic asthmatics. The change in these parameters correlated significantly with the degree of decline in lung function. The interplay between NOD2 signaling, proinflammatory cytokines, CHIT1 activity, heightened oxidative stress and DNA damage orchestrates allergic airway inflammation and thus contributing to the pathogenesis of atopic asthma. These parameters qualified for measurement as part of new noninvasive biomarker panels for monitoring asthma severity. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA Damage Response in Cisplatin-Induced Nephrotoxicity

PubMed Central

Zhu, Shiyao; Pabla, Navjotsingh; Tang, Chengyuan; He, Liyu; Dong, Zheng

2015-01-01

Cisplatin and its derivatives are widely used chemotherapeutic drugs for cancer treatment. However, they have debilitating side-effects in normal tissues and induce ototoxicity, neurotoxicity, and nephrotoxicity. In kidneys, cisplatin preferentially accumulates in renal tubular cells causing tubular cell injury and death, resulting in acute kidney injury (AKI). Recent studies have suggested that DNA damage and the associated DNA damage response (DDR) is an important pathogenic mechanism of AKI following cisplatin treatment. Activation of DDR may lead to cell cycle arrest and DNA repair for cell survival or, in the presence of severe injury, kidney cell death. Modulation of DDR may provide novel renoprotective strategies for cancer patients undergoing cisplatin chemotherapy. PMID:26564230

A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA.

PubMed

Ling, F; Morioka, H; Ohtsuka, E; Shibata, T

2000-12-15

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30 degrees C and shows extensive vegetative petite induction by UV irradiation at 30 degrees C or when cultivated at a higher temperature (37 degrees C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37 degrees C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37 degrees C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.

Impaired mitochondrial Fe-S cluster biogenesis activates the DNA damage response through different signaling mediators.

PubMed

Pijuan, Jordi; María, Carlos; Herrero, Enrique; Bellí, Gemma

2015-12-15

Fe-S cluster biogenesis machinery is required for multiple DNA metabolism processes. In this work, we show that, in Saccharomyces cerevisiae, defects at different stages of the mitochondrial Fe-S cluster assembly machinery (ISC) result in increased spontaneous mutation rate and hyper-recombination, accompanied by an increment in Rad52-associated DNA repair foci and a higher phosphorylated state of γH2A histone, altogether supporting the presence of constitutive DNA lesions. Furthermore, ISC assembly machinery deficiency elicits a DNA damage response that upregulates ribonucleotide reductase activity by promoting the reduction of Sml1 levels and the cytosolic redistribution of Rnr2 and Rnr4 enzyme subunits. Depending on the impaired stage of the ISC machinery, different signaling pathway mediators contribute to such a response, converging on Dun1. Thus, cells lacking the glutaredoxin Grx5, which are compromised at the core ISC system, show Mec1- and Rad53-independent Dun1 activation, whereas both Mec1 and Chk1 are required when the non-core ISC member Iba57 is absent. Grx5-null cells exhibit a strong dependence on the error-free post-replication repair and the homologous recombination pathways, demonstrating that a DNA damage response needs to be activated upon ISC impairment to preserve cell viability. © 2015. Published by The Company of Biologists Ltd.

Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases. PMID:21437275

Chronic Oxidative Damage together with Genome Repair Deficiency in the Neurons is a Double Whammy for Neurodegeneration: Is Damage Response Signaling a Potential Therapeutic Target?

PubMed Central

Wang, Haibo; Dharmalingam, Prakash; Vasquez, Velmarini; Mitra, Joy; Boldogh, Istvan; Rao, K. S.; Kent, Thomas A.; Mitra, Sankar; Hegde, Muralidhar L.

2016-01-01

A foremost challenge for the neurons, which are among the most oxygenated cells, is the genome damage caused by chronic exposure to endogenous reactive oxygen species (ROS), formed as cellular respiratory byproducts. Strong metabolic activity associated with high transcriptional levels in these long lived post-mitotic cells render them vulnerable to oxidative genome damage, including DNA strand breaks and mutagenic base lesions. There is growing evidence for the accumulation of unrepaired DNA lesions in the central nervous system (CNS) during accelerated ageing and progressive neurodegeneration. Several germ line mutations in DNA repair or DNA damage response (DDR) signaling genes are uniquely manifested in the phenotype of neuronal dysfunction and are etiologically linked to many neurodegenerative disorders. Studies in our lab and elsewhere revealed that pro-oxidant metals, ROS and misfolded amyloidogenic proteins not only contribute to genome damage in CNS, but also impede their repair/DDR signaling leading to persistent damage accumulation, a common feature in sporadic neurodegeneration. Here, we have reviewed recent advances in our understanding of the etiological implications of DNA damage vs. repair imbalance, abnormal DDR signaling in triggering neurodegeneration and potential of DDR as a target for the amelioration of neurodegenerative diseases. PMID:27663141

Neighboring base damage induced by permanganate oxidation of 8-oxoguanine in DNA.

PubMed Central

Koizume, S; Inoue, H; Kamiya, H; Ohtsuka, E

1998-01-01

We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G. PMID:9671825

Transcriptional upregulation of p19INK4d upon diverse genotoxic stress is critical for optimal DNA damage response.

PubMed

Ceruti, Julieta M; Scassa, María E; Marazita, Mariela C; Carcagno, Abel C; Sirkin, Pablo F; Cánepa, Eduardo T

2009-06-01

p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.

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

PubMed

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

2016-09-06

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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Anhydrobiosis-Associated Nuclear DNA Damage and Repair in the Sleeping Chironomid: Linkage with Radioresistance

PubMed Central

Vanyagina, Veronica; Malutina, Ludmila; Cornette, Richard; Sakashita, Tetsuya; Hamada, Nobuyuki; Kikawada, Takahiro; Kobayashi, Yasuhiko; Okuda, Takashi

2010-01-01

Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions (4He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3–4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation. PMID:21103355

Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect

PubMed Central

Salvati, Erica; Leonetti, Carlo; Rizzo, Angela; Scarsella, Marco; Mottolese, Marcella; Galati, Rossella; Sperduti, Isabella; Stevens, Malcolm F.G.; D’Incalci, Maurizio; Blasco, Maria; Chiorino, Giovanna; Bauwens, Serge; Horard, Béatrice; Gilson, Eric; Stoppacciaro, Antonella; Zupi, Gabriella; Biroccio, Annamaria

2007-01-01

Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors γ-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA–binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy. PMID:17932567

Comparative analysis of the relative potential of silver, zinc-oxide and titanium-dioxide nanoparticles against UVB-induced DNA damage for the prevention of skin carcinogenesis

PubMed Central

Arora, Sumit; Omar, Yousef; Ijaz, Zohaib Mohammad; AL-Ghadhban, Ahmed; Deshmukh, Sachin K.; Carter, James E.; Singh, Ajay P.; Singh, Seema

2016-01-01

Sunscreen formulations containing UVB filters, such as Zinc-oxide (ZnO) and titanium-dioxide (TiO2) nanoparticles (NPs) have been developed to limit the exposure of human skin to UV-radiations. Unfortunately, these UVB protective agents have failed in controlling the skin cancer incidence. We recently demonstrated that silver nanoparticles (Ag-NPs) could serve as novel protective agents against UVB-radiations. Here our goal was to perform comparative analysis of direct and indirect UVB-protection efficacy of ZnO-, TiO2- and Ag-NPs. Sun-protection-factor calculated based on their UVB-reflective/absorption abilities was the highest for TiO2-NPs followed by Ag- and ZnO-NPs. This was further confirmed by studying indirect protection of UVB radiation-induced death of HaCaT cells. However, only Ag-NPs were active in protecting HaCaT cells against direct UVB-induced DNA-damage by repairing bulky-DNA lesions through nucleotide-excision-repair mechanism. Moreover, Ag-NPs were also effective in protecting HaCaT cells from UVB-induced oxidative DNA damage by enhancing SOD/CAT/GPx activity. In contrast, ZnO- and TiO2-NPs not only failed in providing any direct protection from DNA-damage, but rather enhanced oxidative DNA-damage by increasing ROS production. Together, these findings raise concerns about safety of ZnO- and TiO2-NPs and establish superior protective efficacy of Ag-NPs. PMID:27693632

Lycium barbarum polysaccharide protects human keratinocytes against UVB-induced photo-damage.

PubMed

Li, Huaping; Li, Zhenjie; Peng, Liqian; Jiang, Na; Liu, Qing; Zhang, Erting; Liang, Bihua; Li, Runxiang; Zhu, Huilan

2017-02-01

Ultraviolet B (UVB) irradiation plays a key role in skin damage, which induces oxidative and inflammatory damages, thereby causing photoaging or photocarcinogenesis. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses significant antioxidative and anti-inflammatory effects on multiple tissues. In the present study, the photoprotective effects and potential underlying molecular mechanisms of LBP against UVB-induced photo-damage were investigated in immortalized human keratinocytes (HaCaT cells). The data indicated that pretreatment with LBP significantly attenuated UVB-induced decrease in cell viability, increase in ROS production and DNA damage. LBP also significantly suppressed UVB-induced p38 MAPK activation, and subsequently reversed caspase-3 activation and MMP-9 expression. Notably, LBP was found to induce Nrf2 nuclear translocation and increase the expression of Nrf2-dependent ARE target genes. Furthermore, the protective effects of LBP were abolished by siRNA-mediated Nrf2 silencing. These results showed that the antioxidant LBP could partially protect against UVB irradiation-induced photo-damage through activation of Nrf2/ARE pathway, thereby scavenging ROS and reducing DNA damage, and subsequently suppressing UVB-induced p38 MAP pathway. Thus, LBP can be potentially used for skincare against oxidative damage from environmental insults.

THE ATM-SMC1 PATHWAY IS ESSENTIAL FOR ACTIVATION OF THE CHROMIUM[VI]-INDUCED S-PHASE CHECKPOINT

EPA Science Inventory

Hexavalent chromium (Cr[VI] is a common industrial waste product, an environmental pollutant, and a recognized human carcinogen. Following cellular uptake, Cr[VI] can cause DNA damage, however, the mechanisims by which mammalian cells respond to Cr-induced DNA damage remain to b...

Low dose radiation adaptive protection to control neurodegenerative diseases.

PubMed

Doss, Mohan

2014-05-01

Concerns have been expressed recently regarding the observed increased DNA damage from activities such as thinking and exercise. Such concerns have arisen from an incomplete accounting of the full effects of the increased oxidative damage. When the effects of the induced adaptive protective responses such as increased antioxidants and DNA repair enzymes are taken into consideration, there would be less endogenous DNA damage during the subsequent period of enhanced defenses, resulting in improved health from the thinking and exercise activities. Low dose radiation (LDR), which causes oxidative stress and increased DNA damage, upregulates adaptive protection systems that may decrease diseases in an analogous manner. Though there are ongoing debates regarding LDR's carcinogenicity, with two recent advisory committee reports coming to opposite conclusions, data published since the time of the reports have overwhelmingly ruled out its carcinogenicity, paving the way for consideration of its potential use for disease reduction. LDR adaptive protection is a promising approach to control neurodegenerative diseases, for which there are no methods of prevention or cure. Preparation of a compelling ethics case would pave the way for LDR clinical studies and progress in dealing with neurodegenerative diseases.

Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Fengxia; Zhang, Minjie; University of Chinese Academy of Sciences, Beijing 100049

2014-10-03

Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage.more » Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.« less

Molecular mechanisms by which oxidative DNA damage promotes telomerase activity.

PubMed

Lee, Hui-Ting; Bose, Arindam; Lee, Chun-Ying; Opresko, Patricia L; Myong, Sua

2017-11-16

Telomeres are highly susceptible to oxidative DNA damage, which if left unrepaired can lead to dysregulation of telomere length homeostasis. Here we employed single molecule FRET, single molecule pull-down and biochemical analysis to investigate how the most common oxidative DNA lesions, 8-oxoguanine (8oxoG) and thymine glycol (Tg), regulate the structural properties of telomeric DNA and telomerase extension activity. In contrast to 8oxoG which disrupts the telomeric DNA structure, Tg exhibits substantially reduced perturbation of G-quadruplex folding. As a result, 8oxoG induces high accessibility, whereas Tg retains limited accessibility, of telomeric G-quadruplex DNA to complementary single stranded DNA and to telomere binding protein POT1. Surprisingly, the Tg lesion stimulates telomerase loading and activity to a similar degree as an 8oxoG lesion. We demonstrate that this unexpected stimulation arises from Tg-induced conformational alterations and dynamics in telomeric DNA. Despite impacting structure by different mechanisms, both 8oxoG and Tg enhance telomerase binding and extension activity to the same degree, potentially contributing to oncogenesis. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Error-prone repair and translesion synthesis III: the activation of UmuD (or less is more).

PubMed

Bridges, Bryn A

2005-08-15

Following DNA damage to Escherichia coli bacteria, RecA protein is activated by binding to single stranded DNA and cleaves its own gene repressor (LexA protein). Two papers from Graham Walker's laboratory showed that several bacterial genes in addition to RecA are repressed by the LexA repressor and are inducible following DNA damage [C.J. Keyon, G.C. Walker, DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, in: Proceedings of the National Academy of Sciences of the United States of America 77, 1980, pp. 2819--2823] and predicted that one of them (UmuD) might itself be subject to activation by a further cleavage reaction involving activated RecA protein [K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, G.C. Walker, umuD,C and mucA,B operans whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology, in: Proceedings of the National Academy of Sciences of the United States of America 82, 1985, pp. 4331--4335]. The processed form of UmuD, termed UmuD', later proved to be a subunit of DNA polymerase V, a key enzyme involved in translesion synthesis.

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

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.

Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress.

PubMed

Latour, I; Leunda-Casi, A; Denef, J F; Buc Calderon, P

2000-01-10

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics. Copyright 2000 Academic Press.

Transient spectra study on photo-dynamics of curcumin

NASA Astrophysics Data System (ADS)

Qian, Tingting; Wang, Mei; Wang, Jiao; Zhu, Rongrong; He, Xiaolie; Sun, Xiaoyu; Sun, Dongmei; Wang, Qingxiu; Wang, ShiLong

2016-09-01

A novel mechanism of DNA damage induced by photosensitized curcumin (Cur) was explored using laser flash photolysis, pulse radiolysis and gel electrophoresis. Cur neutral radical (Currad) was confirmed as an identical product in photo-sensitization of Cur by laser flash photolysis and pulse radiolysis. A series of reaction rate constants between Currad and nucleic acid bases/nucleotides were determined by pulse radiolysis. Gel electrophoresis was carried out to investigate damage induced by photosensitized Cur to biologically active DNA. The results indicate that the damage to DNA may be caused by Currad produced from the photosensitization of Cur.

Eccentric localization of catalase to protect chromosomes from oxidative damages during meiotic maturation in mouse oocytes.

PubMed

Park, Yong Seok; You, Seung Yeop; Cho, Sungrae; Jeon, Hyuk-Joon; Lee, Sukchan; Cho, Dong-Hyung; Kim, Jae-Sung; Oh, Jeong Su

2016-09-01

The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes.

Cold atmospheric-pressure plasma induces DNA-protein crosslinks through protein oxidation.

PubMed

Guo, Li; Zhao, Yiming; Liu, Dingxin; Liu, Zhichao; Chen, Chen; Xu, Ruobing; Tian, Miao; Wang, Xiaohua; Chen, Hailan; Kong, Michael G

2018-05-03

Reactive oxygen and nitrogen species (ROS and RNS) generated by cold atmospheric-pressure plasma could damage genomic DNA, although the precise type of these DNA damage induced by plasma are poorly characterized. Understanding plasma-induced DNA damage will help to elucidate the biological effect of plasma and guide the application of plasma in ROS-based therapy. In this study, it was shown that ROS and RNS generated by physical plasma could efficiently induce DNA-protein crosslinks (DPCs) in bacteria, yeast, and human cells. An in vitro assay showed that plasma treatment resulted in the formation of covalent DPCs by activating proteins to crosslink with DNA. Mass spectrometry and hydroperoxide analysis detected oxidation products induced by plasma. DPC formation were alleviated by singlet oxygen scavenger, demonstrating the importance of singlet oxygen in this process. These results suggested the roles of DPC formation in DNA damage induced by plasma, which could improve the understanding of the biological effect of plasma and help to develop a new strategy in plasma-based therapy including infection and cancer therapy.

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

PubMed

Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

2017-09-01

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

DNA damage leads to progressive replicative decline but extends the life span of long-lived mutant animals.

PubMed

Lans, H; Lindvall, J M; Thijssen, K; Karambelas, A E; Cupac, D; Fensgård, O; Jansen, G; Hoeijmakers, J H J; Nilsen, H; Vermeulen, W

2013-12-01

Human-nucleotide-excision repair (NER) deficiency leads to different developmental and segmental progeroid symptoms of which the pathogenesis is only partially understood. To understand the biological impact of accumulating spontaneous DNA damage, we studied the phenotypic consequences of DNA-repair deficiency in Caenorhabditis elegans. We find that DNA damage accumulation does not decrease the adult life span of post-mitotic tissue. Surprisingly, loss of functional ERCC-1/XPF even further extends the life span of long-lived daf-2 mutants, likely through an adaptive activation of stress signaling. Contrariwise, NER deficiency leads to a striking transgenerational decline in replicative capacity and viability of proliferating cells. DNA damage accumulation induces severe, stochastic impairment of development and growth, which is most pronounced in NER mutants that are also impaired in their response to ionizing radiation and inter-strand crosslinks. These results suggest that multiple DNA-repair pathways can protect against replicative decline and indicate that there might be a direct link between the severity of symptoms and the level of DNA-repair deficiency in patients.

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

PubMed Central

Adeyemi, Richard O.

2014-01-01

ABSTRACT The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. IMPORTANCE Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. PMID:24965470

The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-09-01

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Activation of ATM by DNA Damaging Agents

DTIC Science & Technology

2004-09-01

risk for breast cancer . Since many anti-tumor chemotherapeutics used in breast cancer treatment have the capacity to induce DNA DSBs, I have...of a subset of downstream effectors of ATM in two human breast cancer cell lines. Studies are now underway to identify proteins that interact with ATM...implications for the treatment of breast cancer patients harboring mutations in ATM. 14. SUBJECT TERMS 15. NUMBER OF PAGES signal transduction, DNA damage and

Genetic spell-checking: gene editing using single-stranded DNA oligonucleotides.

PubMed

Rivera-Torres, Natalia; Kmiec, Eric B

2016-02-01

Single-stranded oligonucleotides (ssODNs) can be used to direct the exchange of a single nucleotide or the repair of a single base within the coding region of a gene in a process that is known, generically, as gene editing. These molecules are composed of either all DNA residues or a mixture of RNA and DNA bases and utilize inherent metabolic functions to execute the genetic alteration within the context of a chromosome. The mechanism of action of gene editing is now being elucidated as well as an understanding of its regulatory circuitry, work that has been particularly important in establishing a foundation for designing effective gene editing strategies in plants. Double-strand DNA breakage and the activation of the DNA damage response pathway play key roles in determining the frequency with which gene editing activity takes place. Cellular regulators respond to such damage and their action impacts the success or failure of a particular nucleotide exchange reaction. A consequence of such activation is the natural slowing of replication fork progression, which naturally creates a more open chromatin configuration, thereby increasing access of the oligonucleotide to the DNA template. Herein, how critical reaction parameters influence the effectiveness of gene editing is discussed. Functional interrelationships between DNA damage, the activation of DNA response pathways and the stalling of replication forks are presented in detail as potential targets for increasing the frequency of gene editing by ssODNs in plants and plant cells. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Chemical Composition, Antioxidant, DNA Damage Protective, Cytotoxic and Antibacterial Activities of Cyperus rotundus Rhizomes Essential Oil against Foodborne Pathogens

PubMed Central

Hu, Qing-Ping; Cao, Xin-Ming; Hao, Dong-Lin; Zhang, Liang-Liang

2017-01-01

Cyperus rotundus L. (Cyperaceae) is a medicinal herb traditionally used to treat various clinical conditions at home. In this study, chemical composition of Cyperus rotundus rhizomes essential oil, and in vitro antioxidant, DNA damage protective and cytotoxic activities as well as antibacterial activity against foodborne pathogens were investigated. Results showed that α-cyperone (38.46%), cyperene (12.84%) and α-selinene (11.66%) were the major components of the essential oil. The essential oil had an excellent antioxidant activity, the protective effect against DNA damage, and cytotoxic effects on the human neuroblastoma SH-SY5Y cell, as well as antibacterial activity against several foodborne pathogens. These biological activities were dose-dependent, increasing with higher dosage in a certain concentration range. The antibacterial effects of essential oil were greater against Gram-positive bacteria as compared to Gram-negative bacteria, and the antibacterial effects were significantly influenced by incubation time and concentration. These results may provide biological evidence for the practical application of the C. rotundus rhizomes essential oil in food and pharmaceutical industries. PMID:28338066

Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1

PubMed Central

Kanwal, Rajnee; Pandey, Mitali; Bhaskaran, Natarajan; MacLennan, Gregory T; Fu, Pingfu; Ponsky, Lee E; Gupta, Sanjay

2014-01-01

The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P<0.0001) levels of 8-oxo-2′-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r=0.2) with loss of GSTP1 activity (34%; P<0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2O2-mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2O2, these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused re-expression of GSTP1, which protected the cells from H2O2-mediated DNA damage through decreased ROS production compared to non-exposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer. PMID:22833520

Western and Chinese antirheumatic drug-induced T cell apoptotic DNA damage uses different caspase cascades and is independent of Fas/Fas ligand interaction.

PubMed

Lai, J H; Ho, L J; Lu, K C; Chang, D M; Shaio, M F; Han, S H

2001-06-01

Spontaneous or therapeutic induction of T cell apoptosis plays a critical role in establishing transplantation tolerance and maintaining remission of autoimmune diseases. We investigated the mechanisms of apoptosis induced by Chinese and Western antirheumatic drugs (ARDs) in human T cells. We found that hydroxychloroquine, Tripterygium wilfordii hook F, and tetrandrine (Tet), but not methotrexate, at therapeutic concentrations can cause T cell death. In addition, Tet selectively killed T cells, especially activated T cells. Although ARD-induced cytotoxicity was mediated through apoptotic mechanisms, Fas/Fas ligand interaction was not required. We further demonstrated that the processes of phosphatidylserine externalization and DNA damage along the ARD-induced T cell apoptotic pathway could operate independently, and that selective inhibition of DNA damage by caspase inhibitors did not prevent T cells from undergoing cell death. Moreover, we found that Tet- and Tripterygium wilfordii hook F-induced T cell DNA damage required caspase-3 activity, and hydroxychloroquine-induced T cell DNA damage was mediated through a caspase-3- and caspase-8-independent, but Z-Asp-Glu-Val-Asp-fluomethyl ketone-sensitive, signaling pathway. Finally, the observation that ARD-induced activation of caspase-3 in both Fas-sensitive and Fas-resistant Jurkat T cells indicates that Fas/Fas ligand interaction plays no role in ARD-induced T cell apoptosis. Our observations provide new information about the complex apoptotic mechanisms of ARDs, and have implications for combining Western and Chinese ARDs that have different immunomodulatory mechanisms in the therapy of autoimmune diseases and transplantation rejection.

Activation of WIP1 Phosphatase by HTLV-1 Tax Mitigates the Cellular Response to DNA Damage

PubMed Central

Dayaram, Tajhal; Lemoine, Francene J.; Donehower, Lawrence A.; Marriott, Susan J.

2013-01-01

Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G1 phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome. PMID:23405243

DNA damage checkpoint recovery and cancer development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong, E-mail: lsteng@zju.edu.cn

2015-06-10

Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observedmore » to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint recovery pathway plays a role in overriding tumor barrier in tumorigenesis. • Recovery protein dysregulation and human cancer development is correlated.« less

DNA and BSA damage inhibitory activities, and anti-acetylcholinesterase, anti-porcine α-amylase and antioxidant properties of Dolichos lablab beans.

PubMed

Habib, Hosam M; Theuri, Serah W; Kheadr, Ehab; Mohamed, Fedah E

2017-02-22

The underutilized Kenyan variety of Dolichos lablab bean seeds serve as a good source of natural antioxidants, which can probably be effective in reducing the risk of occurrence of several diseases. This study was undertaken for the first time to address the limited knowledge regarding the antioxidant activities of lablab beans. Moreover, their DNA damage inhibitory activity, bovine serum albumin (BSA) damage inhibitory activity, and the inhibition of acetylcholinesterase and porcine α-amylase were also investigated. The antioxidant capacity of Dolichos lablab bean seeds extracted with methanol, water or methanol/water combination was evaluated by the ferric-reducing antioxidant power (FRAP) assay, free radical-scavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO) radical-scavenging assay, and 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Results reported in the present study indicate that water, methanol and water/methanol extracts of lablab bean flour exhibited good antioxidant activity by effectively scavenging various free radicals, such as DPPH, NO, and ABTS radicals. The extracts also exhibited protective effects against DNA and BSA damage and inhibitory effects on porcine α-amylase. Findings of this study suggest that extracts from the lablab bean flour would have potential application in food supplements, and pharmaceutical and cosmetic industries.

Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides.

PubMed

Kobayashi, Kaori; Guilliam, Thomas A; Tsuda, Masataka; Yamamoto, Junpei; Bailey, Laura J; Iwai, Shigenori; Takeda, Shunichi; Doherty, Aidan J; Hirota, Kouji

2016-08-02

PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.

Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs

PubMed Central

Dregalla, Ryan C.; Zhou, Junqing; Idate, Rupa R.; Battaglia, Christine L.R.; Liber, Howard L.; Bailey, Susan M.

2010-01-01

Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated roles of the telomere associated poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) tankyrase 1 in both telomere function and the DNA damage response following exposure to ionizing radiation. Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within telomeres, a phenotype with the potential of accelerating cellular senescence. Additionally, depletion of tankyrase 1 resulted in concomitant and rapid reduction of the nonhomologous end-joining protein DNA-PKcs, while Ku86 and ATM protein levels remained unchanged; DNA-PKcs mRNA levels were also unaffected. We found that the requirement of tankyrase 1 for DNA-PKcs protein stability reflects the necessity of its PARP enzymatic activity. We also demonstrated that depletion of tankyrase 1 resulted in proteasome-mediated DNA-PKcs degradation, explaining the associated defective damage response observed; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberration and telomere fusion. We provide the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity and taken together, identify roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging. PMID:21037379

Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

PubMed

Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

2015-09-30

Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells

PubMed Central

Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N.; Guo, Lei; Mei, Nan

2015-01-01

Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

The Role of Replication in Activation of the DNA Damage Checkpoint

DTIC Science & Technology

2006-03-01

St AD Award Number : W81XWH-04-1-0311 TITLE: The Role of Replication in Activation of the DNA Damage Checkpoint PRINCIPAL INVESTIGATOR: Christopher...Van CONTRACTING ORGANIZATION: Stanford University Stanford, CA 94305 REPORT DATE: March 2006 TYPE OF REPORT: Annual Summary PREPARED FOR: U.S. Army...to comply with a collection of information if it does not display a currently valid OMB control number . PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE

YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage.

PubMed

Ciamporcero, E; Shen, H; Ramakrishnan, S; Yu Ku, S; Chintala, S; Shen, L; Adelaiye, R; Miles, K M; Ullio, C; Pizzimenti, S; Daga, M; Azabdaftari, G; Attwood, K; Johnson, C; Zhang, J; Barrera, G; Pili, R

2016-03-24

Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knockdown sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC.

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

PubMed

Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

2014-06-01

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity.

PubMed

Townsend, Michelle; Peck, Connor; Meng, Wei; Heaton, Matthew; Robison, Richard; O'Neill, Kim

2017-04-01

Glyphosate is a highly used active compound in agriculturally based pesticides. The literature regarding the toxicity of glyphosate to human cells has been highly inconsistent. We studied the resulting DNA damage and cytotoxicity of various glyphosate concentrations on human cells to evaluate DNA damaging potential. Utilizing human Raji cells, DNA damage was quantified using the comet assay, while cytotoxicity was further analyzed using MTT viability assays. Several glyphosate concentrations were assessed, ranging from 15 mM to 0.1 μM. We found that glyphosate treatment is lethal to Raji cells at concentrations above 10 mM, yet has no cytotoxic effects at concentrations at or below 100 μM. Treatment concentrations of 1 mM and 5 mM induce statistically significant DNA damage to Raji cells following 30-60 min of treatment, however, cells show a slow recovery from initial damage and cell viability is unaffected after 2 h. At these same concentrations, cells treated with additional compound did not recover and maintained high levels of DNA damage. While the cytotoxicity of glyphosate appears to be minimal for physiologically relevant concentrations, the compound has a definitive cytotoxic nature in human cells at high concentrations. Our data also suggests a mammalian metabolic pathway for the degradation of glyphosate may be present. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Linking loss of sodium-iodide symporter expression to DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyckesvärd, Madeleine Nordén; Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg; Kapoor, Nirmal

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1 nM) calicheamicin blocked NIS mRNA expression andmore » transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress. - Highlights: • DNA damage inhibits polarized iodide transport in normal thyroid cells. • Down-regulation of NIS expression is mediated by activation of the ATM kinase. • Long-term ATM inhibition also represses NIS-mediated iodide transport. • IGF-1 rescues NIS expression and iodide transport in DNA-damaged cells.« less

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage

PubMed Central

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein–protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage. PMID:24675884

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

PubMed

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

Potentiometric sensing of nuclease activities and oxidative damage of single-stranded DNA using a polycation-sensitive membrane electrode.

PubMed

Ding, Jiawang; Qin, Wei

2013-09-15

A simple, general and label-free potentiometric method to measure nuclease activities and oxidative DNA damage in a homogeneous solution using a polycation-sensitive membrane electrode is reported. Protamine, a linear polyionic species, is used as an indicator to report the cleavage of DNA by nucleases such as restriction and nonspecific nucleases, and the damage of DNA induced by hydroxyl radicals. Measurements can be done with a titration mode or a direct detection mode. For the potentiometric titration mode, the enzymatic cleavage dramatically affects the electrostatical interaction between DNA and protamine and thus shifts the response curve for the potentiometric titration of the DNA with protamine. Under the optimized conditions, the enzyme activities can be sensed potentiometrically with detection limits of 2.7×10(-4)U/µL for S1 nuclease, and of 3.9×10(-4)U/µL for DNase I. For the direct detection mode, a biocomplex between protamine and DNA is used as a substrate. The nuclease of interest cleaves the DNA from the protamine/DNA complex into smaller fragments, so that free protamine is generated and can be detected potentiometrically via the polycation-sensitive membrane electrode. Using a direct measurement, the nuclease activities could be rapidly detected with detection limits of 3.2×10(-4)U/µL for S1 nuclease, and of 4.5×10(-4)U/µL for DNase I. Moreover, the proposed potentiometric assays demonstrate the potential applications in the detection of hydroxyl radicals. It is anticipated that the present potentiometric strategy will provide a promising platform for high-throughput screening of nucleases, reactive oxygen species and the drugs with potential inhibition abilities. Copyright © 2013 Elsevier B.V. All rights reserved.

Antioxidative and antigenotoxic activity of extracts from cosmos (Cosmos bipinnatus) flowers.

PubMed

Jang, In-Cheol; Park, Jae-Hee; Park, Eunju; Park, Hae-Ryong; Lee, Seung-Cheol

2008-12-01

The Cosmos bipinnatus has been used in a traditional herbal remedy for various diseases such as jaundice, intermittent fever, and splenomegaly. The present study describes the preliminary evaluation of antioxidant activities and antigenotoxic effect of Cosmos bipinnatus flowers according to four different colors (white, pink, orange, and violet). The antioxidants properties were evaluated by determining TPC, DPPH RSA, ABTS RSA, and RP. The highest TPC of methanolic CFE (at concentration of 1 mg/ml) showed in violet colored CF (1,013 microM), and IC(50) of DPPH RSA, ABTS RSA, and RP were also the lowest in violet colored CFE with values of 0.61, 1.48, and 0.82 mg/ml, respectively. The antigenotoxic effect of the CFE on DNA damage induced by H(2)O(2) in human leukocytes was evaluated by Comet assay. Pretreatments with CFE produced significant reductions in oxidative DNA damage at the concentration of 500 microg/ml, except for violet colored CFE. The ED(50) value of white colored CFE has shown the highest inhibition (0.40 mg/ml) on H(2)O(2) induced DNA damage, followed by orange > pink > violet color. These results suggested that Cosmos bipinnatus has significant antioxidant activity and protective effect against oxidative DNA damage.

Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest

PubMed Central

Lundholm, L; Hååg, P; Zong, D; Juntti, T; Mörk, B; Lewensohn, R; Viktorsson, K

2013-01-01

Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes. PMID:23370278

Oxidative DNA damage and mammary cell proliferation by alcohol-derived salsolinol.

PubMed

Murata, Mariko; Midorikawa, Kaoru; Kawanishi, Shosuke

2013-10-21

Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

PubMed Central

Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

2016-01-01

Background Diabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration. Methods Female T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals. Results No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group. Conclusion BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical treatment with regular health checks in T2DM patients in Austria. PMID:27598300

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2.

PubMed

Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

2016-01-01

Diabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration. Female T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals. No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group. BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical treatment with regular health checks in T2DM patients in Austria.

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

PubMed Central

Shell, Steven M.; Hawkins, Edward K.; Tsai, Miaw-Sheue; Hlaing, Aye Su; Rizzo, Carmelo J.; Chazin, Walter J.

2013-01-01

The xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results support an indirect read-out model for sensing the presence of lesions by human XPC and suggest XPC may act as a general sensor of damaged DNA capable of recognizing DNA containing lesions not repaired by NER. PMID:24051049

Caffeic acid and quercetin protect erythrocytes against the oxidative stress and the genotoxic effects of lambda-cyhalothrin in vitro.

PubMed

Abdallah, Fatma Ben; Fetoui, H; Fakhfakh, F; Keskes, L

2012-01-01

Lambda-cyhalothrin (LTC) is a synthetic pyrethroid with a broad spectrum of insecticidal and acaricidal activities used to control wide range of insect pests in a variety of applications. The aim of this study was to examine (i) the potency of LTC to induce oxidative stress response in rat erythrocytes in vitro and (ii) the role of caffeic acid (20 μM) and/or quercetin (10 μM) in preventing the cytotoxic effects. Erythrocytes were divided into four portions. The erythrocytes of the first portion were incubated for 4 h at 37°C with different concentrations (0, 50 and 100 μM) of LTC. The others portions were pretreated with caffeic acid and/or quercetin for 30 min prior to LTC incubation. Lipid peroxidation, protein oxidation, antioxidant enzyme activities and DNA damage were examined. LTC at different concentrations causes increased levels of lipid peroxidation, protein oxidation, DNA damage and decreased antioxidant enzyme activities. Combined caffeic acid and quercetin pretreatments significantly reduced the levels of lipid peroxidation markers, that is thiobarbituric acid reactive substance (TBARS), protein carbonyls (PCO) and decreased DNA damage in LTC portion. Further, combined caffeic acid and quercetin pretreatment maintain antioxidant enzyme activities and glutathione content near to normal values. These results suggest that LTC exerts its toxic effect by increasing lipid peroxidation, altering the antioxidant enzyme activities and DNA damage. Caffeic acid and quercetin pretreatments prevent the toxic effects of LTC, suggesting their role as a potential antioxidant.

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers.

PubMed

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-03-28

This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted.

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

PubMed Central

Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

2016-01-01

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins.

PubMed

O'Rourke, Thomas W; Doudican, Nicole A; Mackereth, Melinda D; Doetsch, Paul W; Shadel, Gerald S

2002-06-01

The mitochondrial genome is a significant target of exogenous and endogenous genotoxic agents; however, the determinants that govern this susceptibility and the pathways available to resist mitochondrial DNA (mtDNA) damage are not well characterized. Here we report that oxidative mtDNA damage is elevated in strains lacking Ntg1p, providing the first direct functional evidence that this mitochondrion-localized, base excision repair enzyme functions to protect mtDNA. However, ntg1 null strains did not exhibit a mitochondrial respiration-deficient (petite) phenotype, suggesting that mtDNA damage is negotiated by the cooperative actions of multiple damage resistance pathways. Null mutations in ABF2 or PIF1, two genes implicated in mtDNA maintenance and recombination, exhibit a synthetic-petite phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strains. This phenotype was partially rescued by malonic acid, indicating that reactive oxygen species generated by the electron transport chain contribute to mitochondrial dysfunction in abf2 Delta strains. In contrast, when two other genes involved in mtDNA recombination, CCE1 and NUC1, were inactivated a strong synthetic-petite phenotype was not observed, suggesting that the effects mediated by Abf2p and Pif1p are due to novel activities of these proteins other than recombination. These results document the existence of recombination-independent mechanisms in addition to base excision repair to cope with oxidative mtDNA damage in Saccharomyces cerevisiae. Such systems are likely relevant to those operating in human cells where mtDNA recombination is less prevalent, validating yeast as a model system in which to study these important issues.

Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

PubMed

Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

2016-11-04

Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

Reduced Synchronization Persistence in Neural Networks Derived from Atm-Deficient Mice

PubMed Central

Levine-Small, Noah; Yekutieli, Ziv; Aljadeff, Jonathan; Boccaletti, Stefano; Ben-Jacob, Eshel; Barzilai, Ari

2011-01-01

Many neurodegenerative diseases are characterized by malfunction of the DNA damage response. Therefore, it is important to understand the connection between system level neural network behavior and DNA. Neural networks drawn from genetically engineered animals, interfaced with micro-electrode arrays allowed us to unveil connections between networks’ system level activity properties and such genome instability. We discovered that Atm protein deficiency, which in humans leads to progressive motor impairment, leads to a reduced synchronization persistence compared to wild type synchronization, after chemically imposed DNA damage. Not only do these results suggest a role for DNA stability in neural network activity, they also establish an experimental paradigm for empirically determining the role a gene plays on the behavior of a neural network. PMID:21519382

Expression of the genetic suppressor element 24.2 (GSE24.2) decreases DNA damage and oxidative stress in X-linked dyskeratosis congenita cells.

PubMed

Manguan-Garcia, Cristina; Pintado-Berninches, Laura; Carrillo, Jaime; Machado-Pinilla, Rosario; Sastre, Leandro; Pérez-Quilis, Carme; Esmoris, Isabel; Gimeno, Amparo; García-Giménez, Jose Luis; Pallardó, Federico V; Perona, Rosario

2014-01-01

The predominant X-linked form of Dyskeratosis congenita results from mutations in DKC1, which encodes dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Here we have found that an increased basal and induced DNA damage response occurred in X-DC cells in comparison with normal cells. DNA damage that is also localized in telomeres results in increased heterochromatin formation and senescence. Expression of a cDNA coding for GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized GSE24.2 peptide is able to rescue basal DNA damage in X-DC cells. We have also observed an increase in oxidative stress in X-DC cells and expression of GSE24.2 was able to diminish it. Altogether our data indicated that supplying GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.

Deciphering the Binding between Nupr1 and MSL1 and Their DNA-Repairing Activity

PubMed Central

Doménech, Rosa; Pantoja-Uceda, David; Gironella, Meritxell; Santoro, Jorge; Velázquez-Campoy, Adrián; Neira, José L.; Iovanna, Juan L.

2013-01-01

The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 µM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 µM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 µM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered (“fuzzy”), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners. PMID:24205110

Study of interaction of antimutagenic 1,4-dihydropyridine AV-153-Na with DNA-damaging molecules and its impact on DNA repair activity.

PubMed

Leonova, Elina; Rostoka, Evita; Sauvaigo, Sylvie; Baumane, Larisa; Selga, Turs; Sjakste, Nikolajs

2018-01-01

1,4-dihydropyridines (1,4-DHP) possesses important biochemical and pharmacological properties, including antioxidant and antimutagenic activities. It was shown that the antimutagenic 1,4-dihydropyridine AV-153-Na interacts with DNA. The aim of the current study was to test the capability of the compound to scavenge peroxynitrite and hydroxyl radical, to test intracellular distribution of the compound, and to assess the ability of the compound to modify the activity of DNA repair enzymes and to protect the DNA in living cells against peroxynitrite-induced damage. Peroxynitrite decomposition was assayed by UV spectroscopy, hydroxyl radical scavenging-by EPR spectroscopy. DNA breakage was determined by the "comet method", activity of DNA repair enzymes-using Glyco-SPOT and ExSy-SPOT assays. Intracellular distribution of the compound was studied by laser confocal scanning fluorescence microscopy. Fluorescence spectroscopy titration and circular dichroism spectroscopy were used to study interactions of the compound with human serum albumin. Some ability to scavenge hydroxyl radical by AV-153-Na was detected by the EPR method, but it turned out to be incapable of reacting chemically with peroxynitrite. However, AV-153-Na effectively decreased DNA damage produced by peroxynitrite in cultured HeLa cells. The Glyco-SPOT test essentially revealed an inhibition by AV-153-Na of the enzymes involved thymine glycol repair. Results with ExSy-SPOT chip indicate that AV-153-Na significantly stimulates excision/synthesis repair of 8-oxoguanine (8-oxoG), abasic sites (AP sites) and alkylated bases. Laser confocal scanning fluorescence microscopy demonstrated that within the cells AV-153-Na was found mostly in the cytoplasm; however, a stain in nucleolus was also detected. Binding to cytoplasmic structures might occur due to high affinity of the compound to proteins revealed by spectroscopical methods. Activation of DNA repair enzymes after binding to DNA appears to be the basis for the antimutagenic effects of AV-153-Na.

Ku70 inhibits gemcitabine-induced DNA damage and pancreatic cancer cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Jiali; Hui, Pingping; Meng, Wenying

The current study focused on the role of Ku70, a DNA-dependent protein kinase (DNA-PK) complex protein, in pancreatic cancer cell resistance to gemcitabine. In both established cell lines (Mia-PaCa-2 and PANC-1) and primary human pancreatic cancer cells, shRNA/siRNA-mediated knockdown of Ku70 significantly sensitized gemcitabine-induced cell death and proliferation inhibition. Meanwhile, gemcitabine-induced DNA damage and subsequent pancreatic cancer cell apoptosis were also potentiated with Ku70 knockdown. On the other hand, exogenous overexpression of Ku70 in Mia-PaCa-2 cells suppressed gemcitabine-induced DNA damage and subsequent cell apoptosis. In a severe combined immune deficient (SCID) mice Mia-PaCa-2 xenograft model, gemcitabine-induced anti-tumor activity was remarkably pontificatedmore » when combined with Ku70 shRNA knockdown in the xenografts. The results of this preclinical study imply that Ku70 might be a primary resistance factor of gemcitabine, and Ku70 silence could significantly chemo-sensitize gemcitabine in pancreatic cancer cells. - Highlights: • Ku70 knockdown sensitizes gemcitabine-induced killing of pancreatic cancer cells. • Ku70 knockdown facilitates gemcitabine-induced DNA damage and cell apoptosis. • Ku70 overexpression deceases gemcitabine's sensitivity in pancreatic cancer cells. • Ku70 knockdown sensitizes gemcitabine-induced anti-tumor activity in vivo.« less

TERT regulates telomere-related senescence and apoptosis through DNA damage response in male germ cells exposed to BPDE in vitro and to B[a]P in vivo.

PubMed

Ling, Xi; Yang, Wang; Zou, Peng; Zhang, Guowei; Wang, Zhi; Zhang, Xi; Chen, Hongqiang; Peng, Kaige; Han, Fei; Liu, Jinyi; Cao, Jia; Ao, Lin

2018-04-01

Increasing evidence shows that impaired telomere function is associated with male infertility, and various environmental factors are believed to play a pivotal role in telomerase deficiency and telomere shortening. Benzo[a]pyrene (B[a]P), a ubiquitous pollutant of polycyclic aromatic hydrocarbons (PAHs), can act as a reproductive toxicant; however, the adverse effect of B[a]P on telomeres in male reproductive cells has never been studied, and the related mechanisms remain unclear. In this study, we explored the effects of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), the active metabolite of B[a]P, on telomere dysfunction in mouse spermatocyte-derived cells (GC-2) and also the potential role of telomerase in BPDE-induced spermatogenic cell damage. The results showed that BPDE induced cell viability inhibition, senescence, and apoptosis in GC-2 cells in a dose-dependent manner. Shortened telomeres, telomere-associated DNA damage, reduced telomerase activity, and TERT expression were also observed in BPDE-treated cells, accompanied with the activation of DNA damage response pathway (ATM/Chk1/p53/p21). Moreover, by establishing the TERT knockdown and re-expression cell models, we found that TERT regulated telomere length and the expression of DNA damage response-related proteins to influence senescence and apoptosis in GC-2 cells. These in vitro findings were further confirmed in vivo in the testicular cells of rats orally administrated with B[a]P for 7 days. B[a]P treatment resulted in histological lesions, apoptosis, and senescence in the testes of rats, which were accompanied by shortened telomeres, reduced levels of TERT protein, and increased expression of DNA damage response-related proteins. In conclusion, it can be concluded that TERT-mediated telomere dysfunction contributes to B[a]P- and BPDE-induced senescence and apoptosis through DNA damage response in male reproductive cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2.

PubMed

Xie, Suqing; Wu, Huiyun; Wang, Qi; Kunicki, Jan; Thomas, Raymond O; Hollingsworth, Robert E; Cogswell, John; Dai, Wei

2002-01-01

Polo-like kinase 3 (Plk3, alternatively termed Prk) is involved in the regulation of DNA damage checkpoint as well as in M-phase function. Plk3 physically interacts with p53 and phosphorylates this tumor suppressor protein on serine-20, suggesting that the role of Plk3 in cell cycle progression is mediated, at least in part, through direct regulation of p53. Here we show that Plk3 is rapidly activated by reactive oxygen species in normal diploid fibroblast cells (WI-38), correlating with a subsequent increase in p53 protein level. Plk3 physically interacts with Chk2 and the interaction is enhanced upon DNA damage. In addition, Chk2 immunoprecipitated from cell lysates of Daudi (which expressed little Plk3) is capable of stimulating the kinase activity of purified recombinant Plk3 in vitro, and this stimulation is more pronounced when Plk3 is supplemented with Chk2 immunoprecipitated from Daudi after DNA damage. Furthermore, ectopic expression Chk2 activates cellular Plk3. Together, our studies suggest Chk2 may mediate direct activation of Plk3 in response to genotoxic stresses.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

PubMed

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

PubMed Central

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

Replication of damaged DNA in vitro is blocked by p53

PubMed Central

Zhou, Jianmin; Prives, Carol

2003-01-01

The tumor suppressor protein p53 may have other roles and functions in addition to its well-documented ability to serve as a sequence-specific transcriptional activator in response to DNA damage. We showed previously that p53 can block the replication of polyomavirus origin-containing DNA (Py ori-DNA) in vitro when p53 binding sites are present on the late side of the Py ori. Here we have both further extended these observations and have also examined whether p53 might be able to bind directly to and inhibit the replication of damaged DNA. We found that p53 strongly inhibits replication of γ-irradiated Py ori-DNA and such inhibition requires both the central DNA binding domain and the extreme C-terminus of the p53 protein. An endogenous p53 binding site lies within the Py origin and is required for the ability of p53 to block initiation of replication from γ-irradiated Py ori-DNA, suggesting the possibility of DNA looping caused by p53 binding both non-specifically to sites of DNA damage and specifically to the endogenous site in the polyomavirus origin. Our results thus suggest the possibility that under some circumstances p53 might serve as a direct regulator of DNA replication and suggest as well an additional function for cooperation between its two autonomous DNA binding domains. PMID:12853603

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

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

PubMed

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.

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

PubMed

Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

2015-07-09

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

PubMed Central

Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

2015-01-01

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

Uridine homeostatic disorder leads to DNA damage and tumorigenesis.

PubMed

Cao, Zhe; Ma, Jun; Chen, Xinchun; Zhou, Boping; Cai, Chuan; Huang, Dan; Zhang, Xuewen; Cao, Deliang

2016-03-28

Uridine is a natural nucleoside precursor of uridine monophosphate in organisms and thus is considered to be safe and is used in a wide range of clinical settings. The far-reaching effects of pharmacological uridine have long been neglected. Here, we report that the homeostatic disorder of uridine is carcinogenic. Targeted disruption (-/-) of murine uridine phosphorylase (UPase) disrupted the homeostasis of uridine and increased spontaneous tumorigenesis by more than 3-fold. Multiple tumors (e.g., lymphoma, hepatoma and lung adenoma) occurred simultaneously in some UPase deficient mice, but not in wild-type mice raised under the same conditions. In the tissue from UPase -/- mice, the 2'-deoxyuridine,5'-triphosphate (dUTP) levels and uracil DNA were increased and p53 was activated with an increased phospho-Ser18 p53 level. Exposing cell lines (e.g., MCF-7, RKO, HCT-8 and NCI-H460) to uridine (10 or 30 µM) led to uracil DNA damage and p53 activation, which in turn triggered the DNA damage response. In these cells, phospho-ATM, phospho-CHK2, and phospho-γH2AX were increased by uridine. These data suggest that uridine homeostatic disorder leads to uracil DNA damage and that pharmacological uridine may be carcinogenic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Inhibition of cell signaling by the combi-nitrosourea FD137 in the androgen independent DU145 prostate cancer cell line.

PubMed

Qiu, Qiyu; Dudouit, Fabienne; Banerjee, Ranjita; McNamee, James P; Jean-Claude, Bertrand J

2004-04-01

FD137, a nitrosourea appended to a quinazoline ring, was designed to simultaneously block epidermal growth factor receptor (EGFR)-mediated signaling and damage genomic DNA in refractory EGF-dependent prostate tumors. The mixed inhibition of cell signaling and DNA damage by FD137 were determined by Western blotting, RT-PCR, flow cytometry, sulforhodamine B (SRB), and comet assay. FD137 and its metabolite FD110 induced a dose-dependent increase in inhibition of EGF-stimulated EGFR autophosphorylation and this translated into blockade of c-fos gene expression in DU145 cells. FD137 induced significant levels of DNA damage and showed 150-fold greater anti-proliferative activity than BCNU, a classical nitrosourea. In contrast to BCNU, complete inhibition of EGF-induced cell transition to S-phase was observed at concentrations of FD137 as low as 3 microM. FD137 could not only damage DNA, but also significantly block downstream EGFR-mediated signaling. The superior activity of FD137 may be imputable to the combined effect of its mixed EGFR/DNA targeting properties. This novel strategy may well represent a new approach to target nitrosoureas to EGFR-overexpressing carcinomas of the prostate. Copyright 2004 Wiley-Liss, Inc.

Selective antitumor activity of roscovitine in head and neck cancer

PubMed Central

Biktasova, Asel; Bellinger, Gary; Yarbrough, Wendell G.; Issaeva, Natalia

2016-01-01

Radiation and chemotherapy that are commonly used to treat human cancers damage cellular DNA. DNA damage appears to be more toxic to cancer cells than normal cells, most likely due to deregulated checkpoint activation and/or deficiency in DNA repair pathways that are characteristics of many tumors. However, unwanted side effects arise as a result of DNA damage to normal cells during the treatment. Here, we show that roscovitine, a cyclin-dependent kinase (CDK) inhibitor that inhibits CDK-1, CDK-2, CDK-5, CDK-7, and CDK-9 due to competitive binding to the ATP site on the kinases, causes significant DNA damage followed by p53-dependent cell death in human papilloma virus (HPV)-positive, but not in HPV-negative, head and neck cancer cells. Since HPV positivity was a molecular marker for increased sensitivity of cells to roscovitine, we reasoned that systemic roscovitine administration would not be toxic to healthy HPV-negative tissue. Indeed, low roscovitine doses significantly inhibited the growth of HPV-associated xenografted tumors in mice without causing any detectable side effects. Given that inhibition of CDKs has been shown to inhibit replication of several viruses, we suggest that roscovitine treatment may represent a selective and safe targeted therapeutic option against HPV-positive head and neck cancer. PMID:27233076

XRN2 Links Transcription Termination to DNA Damage and Replication Stress

PubMed Central

Patidar, Praveen L.; Motea, Edward A.; Dang, Tuyen T.; Manley, James L.

2016-01-01

XRN2 is a 5’-3’ exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability. PMID:27437695

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

PubMed

Morales, Julio C; Richard, Patricia; Patidar, Praveen L; Motea, Edward A; Dang, Tuyen T; Manley, James L; Boothman, David A

2016-07-01

XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

Absence of ERK5/MAPK7 delays tumorigenesis in Atm-/- mice.

PubMed

Granados-Jaén, Alba; Angulo-Ibáñez, Maria; Rovira-Clavé, Xavier; Gamez, Celina Paola Vasquez; Soriano, Francesc X; Reina, Manuel; Espel, Enric

2016-11-15

Ataxia-telangiectasia mutated (ATM) is a cell cycle checkpoint kinase that upon activation by DNA damage leads to cell cycle arrest and DNA repair or apoptosis. The absence of Atm or the occurrence of loss-of-function mutations in Atm predisposes to tumorigenesis. MAPK7 has been implicated in numerous types of cancer with pro-survival and pro-growth roles in tumor cells, but its functional relation with tumor suppressors is not clear. In this study, we show that absence of MAPK7 delays death due to spontaneous tumor development in Atm-/- mice. Compared with Atm-/- thymocytes, Mapk7-/-Atm-/- thymocytes exhibited an improved response to DNA damage (increased phosphorylation of H2AX) and a restored apoptotic response after treatment of mice with ionizing radiation. These findings define an antagonistic function of ATM and MAPK7 in the thymocyte response to DNA damage, and suggest that the lack of MAPK7 inhibits thymic lymphoma growth in Atm-/- mice by partially restoring the DNA damage response in thymocytes.

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

PubMed

Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

2016-07-26

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

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

Prevention of DNA damage and anticarcinogenic activity of Activia® in a preclinical model.

PubMed

Limeiras, S M A; Ogo, F M; Genez, L A L; Carreira, C M; Oliveira, E J T; Pessatto, L R; Neves, S C; Pesarini, J R; Schweich, L C; Silva, R A; Cantero, W B; Antoniolli-Silva, A C M B; Oliveira, R J

2017-03-22

Colorectal cancer is a global public health issue. Studies have pointed to the protective effect of probiotics on colorectal carcinogenesis. Activia ® is a lacto probiotic product that is widely consumed all over the world and its beneficial properties are related, mainly, to the lineage of traditional yoghurt bacteria combined with a specific bacillus, DanRegularis, which gives the product a proven capacity to intestinal regulation in humans. The aim of this study was to evaluate the antigenotoxic, antimutagenic, and anticarcinogenic proprieties of the Activia product, in response to damage caused by 1,2-dimethylhydrazine (DMH) in Swiss mice. Activia does not have shown antigenotoxic activity. However, the percent of DNA damage reduction, evaluated by the antimutagenicity assay, ranged from 69.23 to 96.15% indicating effective chemopreventive action. Activia reduced up to 79.82% the induction of aberrant crypt foci by DMH. Facing the results, it is inferred that Activia facilitates the weight loss, prevents DNA damage and pre-cancerous lesions in the intestinal mucosa.

Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Quantitative and Dynamic Imaging of ATM Kinase Activity.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

How Alterations in the Cdt1 Expression Lead to Gene Amplification in Breast Cancer

DTIC Science & Technology

2011-07-01

absence of extrinsic DNA damage. We measured the TLS activity by measuring the mutation frequency in a supF gene (in a shuttle vector) subjected to UV...induced DNA damage before its introduction into the cells. Error-prone TLS activity will mutate the supF gene , which is scored by a blue-white colony...Figure 4A). Sequencing of the mutant supF genes , revealed a mutation spectrum consistent with error prone TLS (Supplemental Table 1). Significantly

Lipogenic-associated gene activity of adipose tissue from beef heifers and relation to production and reproductive traits

USDA-ARS?s Scientific Manuscript database

Transition from adolescence to puberty is marked by changes in metabolic activity. Fatty acid synthase (FASN) catalyzes the de novo synthesis of fatty acids and increased expression has been linked to excess energy intake and obesity. The enzyme, DNA-protein kinase (DNA-PK) plays a role in DNA damag...

Comparative analysis of the relative potential of silver, Zinc-oxide and titanium-dioxide nanoparticles against UVB-induced DNA damage for the prevention of skin carcinogenesis.

PubMed

Tyagi, Nikhil; Srivastava, Sanjeev K; Arora, Sumit; Omar, Yousef; Ijaz, Zohaib Mohammad; Al-Ghadhban, Ahmed; Deshmukh, Sachin K; Carter, James E; Singh, Ajay P; Singh, Seema

2016-12-01

Sunscreen formulations containing UVB filters, such as Zinc-oxide (ZnO) and titanium-dioxide (TiO 2 ) nanoparticles (NPs) have been developed to limit the exposure of human skin to UV-radiations. Unfortunately, these UVB protective agents have failed in controlling the skin cancer incidence. We recently demonstrated that silver nanoparticles (Ag-NPs) could serve as novel protective agents against UVB-radiations. Here our goal was to perform comparative analysis of direct and indirect UVB-protection efficacy of ZnO-, TiO 2 - and Ag-NPs. Sun-protection-factor calculated based on their UVB-reflective/absorption abilities was the highest for TiO 2 -NPs followed by Ag- and ZnO-NPs. This was further confirmed by studying indirect protection of UVB radiation-induced death of HaCaT cells. However, only Ag-NPs were active in protecting HaCaT cells against direct UVB-induced DNA-damage by repairing bulky-DNA lesions through nucleotide-excision-repair mechanism. Moreover, Ag-NPs were also effective in protecting HaCaT cells from UVB-induced oxidative DNA damage by enhancing SOD/CAT/GPx activity. In contrast, ZnO- and TiO 2 -NPs not only failed in providing any direct protection from DNA-damage, but rather enhanced oxidative DNA-damage by increasing ROS production. Together, these findings raise concerns about safety of ZnO- and TiO 2 -NPs and establish superior protective efficacy of Ag-NPs. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way.

PubMed

Kulms, D; Pöppelmann, B; Schwarz, T

2000-05-19

Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.

Multifunctional Ebselen drug functions through the activation of DNA damage response and alterations in nuclear proteins.

PubMed

Azad, Gajendra K; Balkrishna, Shah Jaimin; Sathish, Narayanan; Kumar, Sangit; Tomar, Raghuvir S

2012-01-15

Several studies have demonstrated that Ebselen is an anti-inflammatory and anti-oxidative agent. Contrary to this, studies have also shown a high degree of cellular toxicity associated with Ebselen usage, the underlying mechanism of which remains less understood. In this study we have attempted to identify a possible molecular mechanism behind the above by investigating the effects of Ebselen on Saccharomyces cerevisiae. Significant growth arrest was documented in yeast cells exposed to Ebselen similar to that seen in presence of DNA damaging agents (including methyl methane sulfonate [MMS] and hydroxy urea [HU]). Furthermore, mutations in specific lysine residues in the histone H3 tail (H3 K56R) resulted in increased sensitivity of yeast cells to Ebselen presumably due to alterations in post-translational modifications of histone proteins towards regulating replication and DNA damage repair. Our findings suggest that Ebselen functions through activation of DNA damage response, alterations in histone modifications, activation of checkpoint kinase pathway and derepression of ribonucleotide reductases (DNA repair genes) which to the best of our knowledge is being reported for the first time. Interestingly subsequent to Ebselen exposure there were changes in global yeast protein expression and specific histone modifications, identification of which is expected to reveal a fundamental cellular mechanism underlying the action of Ebselen. Taken together these observations will help to redesign Ebselen-based therapy in clinical trials. Copyright © 2011 Elsevier Inc. All rights reserved.

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

PubMed Central

Vallerga, María Belén; Mansilla, Sabrina F.; Federico, María Belén; Bertolin, Agustina P.; Gottifredi, Vanesa

2015-01-01

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli.

PubMed

Moore, Jessica M; Correa, Raul; Rosenberg, Susan M; Hastings, P J

2017-07-01

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli

PubMed Central

Moore, Jessica M.; Correa, Raul; Rosenberg, Susan M.

2017-01-01

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators. PMID:28727736

DNA Damage Response Genes and the Development of Cancer Metastasis

PubMed Central

Broustas, Constantinos G.; Lieberman, Howard B.

2014-01-01

DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-) factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genomewide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo. PMID:24397478

Mechanism underlying the effect of long-term exposure to low dose of pesticides on DNA integrity.

PubMed

Alleva, Renata; Manzella, Nicola; Gaetani, Simona; Bacchetti, Tiziana; Bracci, Massimo; Ciarapica, Veronica; Monaco, Federica; Borghi, Battista; Amati, Monica; Ferretti, Gianna; Tomasetti, Marco

2018-04-01

Pesticides, including herbicides, insecticides and fungicides, are widely used in intensive agriculture. Recently, the long-term effects of pesticide exposure were found to be associated with many diseases. In this study, we evaluated the long-term effect of low-level exposure to a mixture of pesticides on DNA damage response (DDR) in relation to individual detoxifying variability. A residential population chronically exposed to pesticides was enrolled, biological/environmental pesticide levels; paroxonase 1 (PON-1) activity and 192 Q/R polymorphism and DDR were evaluated at three different periods of pesticide exposure. OGG1-dependent DNA repair activity was decreased in relation to pesticide exposure. The increase of DNA lesions and pesticide levels in the intensive pesticide-spraying period was independent on PON-1 activity. Next, human bronchial epithelial and neuronal cells were used as a model for in vitro evaluation of the mechanistic effect of pesticides. Pesticides induced mitochondrial dysfunction leading to ROS formation. ROS from mitochondria induced DNA damage, which in turn induced OGG1-dependent DNA repair activity through 8-oxoguanine DNA glycosylase 1 (OGG1) expression and activation. Even though OGG1 was overexpressed, an inhibition of its activity, associated with DNA lesion accumulation, was found at prolonged pesticide-exposure. A post-translational regulation of OGG1 by pesticide may be postulated. Taken together, long-term exposure to low-levels of pesticides affects DDR resulting in accumulation of DNA lesions that eventually may lead to cancer or neurological disorders. © 2018 Wiley Periodicals, Inc.

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy

PubMed Central

Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog

2016-01-01

Abstract Aims: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. Results: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. Innovation: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. Conclusion: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072–1083. PMID:26935406

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

PubMed

Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog; Jurkunas, Ula V

2016-06-20

Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.

Microcystin-LR induced DNA damage in human peripheral blood lymphocytes.

PubMed

Zegura, B; Gajski, G; Straser, A; Garaj-Vrhovac, V; Filipič, M

2011-12-24

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

PubMed

Makarova, A V; Kulbachinskiy, A V

2012-06-01

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

Unraveling a mechanism of honey antibacterial action: polyphenol/H₂O₂-induced oxidative effect on bacterial cell growth and on DNA degradation.

PubMed

Brudzynski, Katrina; Abubaker, Kamal; Miotto, Danielle

2012-07-15

Several compounds with antibacterial activities were identified in honey however, a mechanism by which they lead to bacterial growth inhibition and bacterial death remains still unknown. We recently found that honeys possess DNA degrading activity mediated by honey hydrogen peroxide and an unknown honey component(s). Here we provide evidence that active honeys (MIC90 of 6.25-12.5% v/v) possessed significantly higher levels of phenolics (p<0.02) of higher radical scavenging activities (p<0.005) than honeys of average activity. Removal of H2O2 by catalase eliminated bacteriostatic activities caused by both phenolics and H2O2 suggesting that the growth inhibition resulted from the coupling chemistry between these compounds. Both phenolics and H2O2 were involved in DNA degradation by honeys. Treatment of plasmid DNA with H2O2 alone did not affect the DNA integrity but H2O2 removal from honey by catalase prevented DNA degradation. Polyphenols extracted from honeys degraded plasmid DNA in the presence of H2O2 and Cu(II) in the Fenton-type reaction. The extent of DNA degradation was inversely related to the polyphenol concentration in this system as well as in honeys. At low content, honey polyphenols exerted pro-oxidant activity damaging to DNA. In conclusion, honey phenolics with pro-oxidant activities were necessary intermediates that conferred oxidative action of H2O2. Phenolic/H2O2-induced oxidative stress constituted the mechanism of honey bacteriostatic and DNA damaging activities. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

PubMed Central

SOLANKY, DIPESH; HAYDEL, SHELLEY E.

2012-01-01

This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair

PubMed Central

Wienholz, Franziska; Vermeulen, Wim

2017-01-01

Abstract Nucleotide excision repair (NER) comprises two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which remove a wide variety of helix-distorting lesions including UV-induced damage. During NER, a short stretch of single-stranded DNA containing damage is excised and the resulting gap is filled by DNA synthesis in a process called unscheduled DNA synthesis (UDS). UDS is measured by quantifying the incorporation of nucleotide analogues into repair patches to provide a measure of NER activity. However, this assay is unable to quantitatively determine TC-NER activity due to the low contribution of TC-NER to the overall NER activity. Therefore, we developed a user-friendly, fluorescence-based single-cell assay to measure TC-NER activity. We combined the UDS assay with tyramide-based signal amplification to greatly increase the UDS signal, thereby allowing UDS to be quantified at low UV doses, as well as DNA-repair synthesis of other excision-based repair mechanisms such as base excision repair and mismatch repair. Importantly, we demonstrated that the amplified UDS is sufficiently sensitive to quantify TC-NER-derived repair synthesis in GG-NER-deficient cells. This assay is important as a diagnostic tool for NER-related disorders and as a research tool for obtaining new insights into the mechanism and regulation of excision repair. PMID:28088761

C/EBPbeta represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19(Arf).

PubMed

Ewing, S J; Zhu, S; Zhu, F; House, J S; Smart, R C

2008-11-01

CCAAT/enhancer-binding protein-beta (C/EBPbeta) is a mediator of cell survival and tumorigenesis. When C/EBPbeta(-/-) mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19(Arf) and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19(Arf) is dramatically elevated in C/EBPbeta(-/-) epidermis and that C/EBPbeta represses a p19(Arf) promoter reporter. To determine whether p19(Arf) is responsible for the proapoptotic phenotype in C/EBPbeta(-/-) mice, C/EBPbeta(-/-);p19(Arf-/-) mice were generated. C/EBPbeta(-/-);p19(Arf-/-) mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19(Arf) is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPbeta(-/-) epidermis, we generated K14-ER:Ras;C/EBPbeta(-/-) mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPbeta(-/-) mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPbeta represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPbeta may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.

C/EBPβ represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19Arf

PubMed Central

Ewing, SJ; Zhu, S; Zhu, F; House, JS; Smart, RC

2013-01-01

CCAAT/enhancer-binding protein-β (C/EBPβ) is a mediator of cell survival and tumorigenesis. When C/EBPβ−/− mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19Arf and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19Arf is dramatically elevated in C/EBPβ−/− epidermis and that C/EBPβ represses a p19Arf promoter reporter. To determine whether p19Arf is responsible for the proapoptotic phenotype in C/EBPβ−/− mice, C/EBPβ−/−;p19Arf−/− mice were generated. C/EBPβ−/−;p19Arf−/− mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19Arf is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPβ−/− epidermis, we generated K14-ER:Ras; C/EBPβ−/− mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPβ−/− mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPβ represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPβ may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents. PMID:18636078

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

PubMed Central

Zucca, Elisa; Bertoletti, Federica; Wimmer, Ursula; Ferrari, Elena; Mazzini, Giuliano; Khoronenkova, Svetlana; Grosse, Nicole; van Loon, Barbara; Dianov, Grigory; Hübscher, Ulrich; Maga, Giovanni

2013-01-01

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells. PMID:23118481

Oxidative DNA damage and oxidative stress in lead-exposed workers.

PubMed

Dobrakowski, M; Pawlas, N; Kasperczyk, A; Kozłowska, A; Olewińska, E; Machoń-Grecka, A; Kasperczyk, S

2017-07-01

There are many discrepancies among the results of studies on the genotoxicity of lead. The aim of the study was to explore lead-induced DNA damage, including oxidative damage, in relation to oxidative stress intensity parameters and the antioxidant defense system in human leukocytes. The study population consisted of 100 male workers exposed to lead. According to the blood lead (PbB) levels, they were divided into the following three subgroups: a group with PbB of 20-35 μg/dL (low exposure to lead (LE) group), a group with a PbB of 35-50 µg/dL (medium exposure to lead (ME) group), and a group with a PbB of >50 μg/dL (high exposure to lead (HE) group). The control group consisted of 42 healthy males environmentally exposed to lead (PbB < 10 μg/dL). A comet assay was used to measure the DNA damage in leukocytes. We measured the activity of superoxide dismutase (SOD), catalase, glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), and glutathione-S-transferase (GST) as well as the concentration of malondialdehyde (MDA), and the value of the total antioxidant capacity. The level of PbB was significantly higher in the examined subgroups than in the control group. The percentage of DNA in the tail was significantly higher in the LE, ME, and HE subgroups than in the control group by 10% ( p = 0.001), 15% ( p < 0.001), and 20% ( p < 0.001), respectively. The activity of GR was significantly lower in the LE and ME subgroups than in the control group by 25% ( p = 0.007) and 17% ( p = 0.028), respectively. The activity of G6PD was significantly lower in the ME subgroup by 25% ( p = 0.022), whereas the activity of GST was significantly higher in the HE subgroup by 101% ( p = 0.001) than in the control group. Similarly, the activity of SOD was significantly higher in the LE and ME subgroups by 48% ( p = 0.026) and 34% ( p = 0.002), respectively. The concentration of MDA was significantly higher in the LE, ME, and HE subgroups than in the control group by 43% ( p = 0.016), 57% ( p < 0.001), and 108% ( p < 0.001), respectively. Occupational lead exposure induces DNA damage, including oxidative damage, in human leukocytes. The increase in DNA damage was accompanied by an elevated intensity of oxidative stress.

Sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation will increase in lipopolysaccharide-induced inflammation in vitro model.

PubMed

Zuo, Wen-Qi; Hu, Yu-Juan; Yang, Yang; Zhao, Xue-Yan; Zhang, Yuan-Yuan; Kong, Wen; Kong, Wei-Jia

2015-05-29

With the increasing popularity of mobile phones, the potential hazards of radiofrequency electromagnetic radiation (RF-EMR) on the auditory system remain unclear. Apart from RF-EMR, humans are also exposed to various physical and chemical factors. We established a lipopolysaccharide (LPS)-induced inflammation in vitro model to investigate whether the possible sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation (at specific absorption rates: 2, 4 W/kg) will increase. Spiral ganglion neurons (SGN) were obtained from neonatal (1- to 3-day-old) Sprague Dawley® (SD) rats. After the SGN were treated with different concentrations (0, 20, 40, 50, 100, 200, and 400 μg/ml) of LPS, the Cell Counting Kit-8 (CCK-8) and alkaline comet assay were used to quantify cellular activity and DNA damage, respectively. The SGN were treated with the moderate LPS concentrations before RF-EMR exposure. After 24 h intermittent exposure at an absorption rate of 2 and 4 W/kg, DNA damage was examined by alkaline comet assay, ultrastructure changes were detected by transmission electron microscopy, and expression of the autophagy markers LC3-II and Beclin1 were examined by immunofluorescence and confocal laser scanning microscopy. Reactive oxygen species (ROS) production was quantified by the dichlorofluorescin-diacetate assay. LPS (100 μg/ml) induced DNA damage and suppressed cellular activity (P < 0.05). LPS (40 μg/ml) did not exhibit cellular activity changes or DNA damage (P > 0.05); therefore, 40 μg/ml was used to pretreat the concentration before exposure to RF-EMR. RF-EMR could not directly induce DNA damage. However, the 4 W/kg combined with LPS (40 μg/ml) group showed mitochondria vacuoles, karyopyknosis, presence of lysosomes and autophagosome, and increasing expression of LC3-II and Beclin1. The ROS values significantly increased in the 4 W/kg exposure, 4 W/kg combined with LPS (40 μg/ml) exposure, and H2O2 groups (P < 0.05, 0.01). Short-term exposure to radiofrequency electromagnetic radiation could not directly induce DNA damage in normal spiral ganglion neurons, but it could cause the changes of cellular ultrastructure at special SAR 4.0 W/kg when cells are in fragile or micro-damaged condition. It seems that the sensitivity of SGN to damage caused by mobile phone electromagnetic radiation will increase in a lipopolysaccharide-induced inflammation in vitro model.

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

PubMed Central

Kuzminov, Andrei

1999-01-01

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

DNA damage as a biomarker for assessing the effects of suspended solids on the orange-spotted grouper, Epinephelus coioides.

PubMed

Tse, C Y; Chan, K M; Wong, C K

2010-06-01

In Hong Kong, suspended solids (SS) introduced by dredging and mud disposal activities are a major cause of mass mortality in cage-cultured marine fish. We have used DNA damage in liver cells, as determined by the comet assay, to assess the impact of SS on the orange-spotted grouper Epinephelus coioides. Seabed sediments were collected from a heavily polluted site in Victoria Harbor and two less polluted sites in Port Shelter and Mirs Bay. Sediments from Victoria Harbor contained higher levels of copper (Cu) and polycyclic aromatic hydrocarbons (PAHs) than those from the other sites. In a 10-day experiment, SS from all three sites induced significant increase in comet tail length, but not in percentage (%) tail DNA. In a 20-day experiment, fish exposed to polluted SS from Victoria Harbor exhibited a significant increase in comet tail length after 5 days and % tail DNA after 10 days. After a 10-day recovery period, however, DNA damage was reduced as tail length and % tail DNA returned to control levels. These results suggest that DNA damage measured by the comet assay is a highly sensitive biomarker for assessing the genotoxic effects of SS to marine fish.

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

PubMed

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

2015-05-19

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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma.

PubMed

Li, Zhigang; Fan, Erica K; Liu, Jinghua; Scott, Melanie J; Li, Yuehua; Li, Song; Xie, Wen; Billiar, Timothy R; Wilson, Mark A; Jiang, Yong; Wang, Ping; Fan, Jie

2017-05-11

Trauma is a major cause of systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Macrophages (Mφ) direct trauma-induced inflammation, and Mφ death critically influences the progression of the inflammatory response. In the current study, we explored an important role of trauma in inducing mitochondrial DNA (mtDNA) damage in Mφ and the subsequent regulation of Mφ death. Using an animal pseudo-fracture trauma model, we demonstrated that tissue damage induced NADPH oxidase activation and increased the release of reactive oxygen species via cold-inducible RNA-binding protein (CIRP)-TLR4-MyD88 signaling. This in turn, activates endonuclease G, which serves as an executor for the fragmentation of mtDNA in Mφ. We further showed that fragmented mtDNA triggered both p62-related autophagy and necroptosis in Mφ. However, autophagy activation also suppressed Mφ necroptosis and pro-inflammatory responses. This study demonstrates a previously unidentified intracellular regulation of Mφ homeostasis in response to trauma.

Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma

PubMed Central

Li, Zhigang; Fan, Erica K; Liu, Jinghua; Scott, Melanie J; Li, Yuehua; Li, Song; Xie, Wen; Billiar, Timothy R; Wilson, Mark A; Jiang, Yong; Wang, Ping; Fan, Jie

2017-01-01

Trauma is a major cause of systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Macrophages (Mϕ) direct trauma-induced inflammation, and Mϕ death critically influences the progression of the inflammatory response. In the current study, we explored an important role of trauma in inducing mitochondrial DNA (mtDNA) damage in Mϕ and the subsequent regulation of Mϕ death. Using an animal pseudo-fracture trauma model, we demonstrated that tissue damage induced NADPH oxidase activation and increased the release of reactive oxygen species via cold-inducible RNA-binding protein (CIRP)–TLR4–MyD88 signaling. This in turn, activates endonuclease G, which serves as an executor for the fragmentation of mtDNA in Mϕ. We further showed that fragmented mtDNA triggered both p62-related autophagy and necroptosis in Mϕ. However, autophagy activation also suppressed Mϕ necroptosis and pro-inflammatory responses. This study demonstrates a previously unidentified intracellular regulation of Mϕ homeostasis in response to trauma. PMID:28492546

Activation of DNA Damage Repair Pathways by Murine Polyomavirus

PubMed Central

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L.

2016-01-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. PMID:27529739

Wavelength dependence of biological damage induced by UV radiation on bacteria.

PubMed

Santos, Ana L; Oliveira, Vanessa; Baptista, Inês; Henriques, Isabel; Gomes, Newton C M; Almeida, Adelaide; Correia, António; Cunha, Ângela

2013-01-01

The biological effects of UV radiation of different wavelengths (UVA, UVB and UVC) were assessed in nine bacterial isolates displaying different UV sensitivities. Biological effects (survival and activity) and molecular markers of oxidative stress [DNA strand breakage (DSB), generation of reactive oxygen species (ROS), oxidative damage to proteins and lipids, and the activity of antioxidant enzymes catalase and superoxide dismutase] were quantified and statistically analyzed in order to identify the major determinants of cell inactivation under the different spectral regions. Survival and activity followed a clear wavelength dependence, being highest under UVA and lowest under UVC. The generation of ROS, as well as protein and lipid oxidation, followed the same pattern. DNA damage (DSB) showed the inverse trend. Multiple stepwise regression analysis revealed that survival under UVA, UVB and UVC wavelengths was best explained by DSB, oxidative damage to lipids, and intracellular ROS levels, respectively.

Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

PubMed

Fox, Candace R; Parks, Griffith D

2018-04-01

A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI - ) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI - infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI - persistently infected (PI) cells. P/V-CPI - PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI - as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors. IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked to alterations to DNA damage signaling pathways that balance cell survival versus death. Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish persistent infection, the repurposing of drugs that target cellular IAPs as antivirals, and the combined use of DNA-damaging chemotherapy agents in conjunction with oncolytic RNA virus vectors. Copyright © 2018 American Society for Microbiology.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 eventmore » 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.« less

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA.

PubMed

Scheibye-Knudsen, Morten; Tseng, Anne; Borch Jensen, Martin; Scheibye-Alsing, Karsten; Fang, Evandro Fei; Iyama, Teruaki; Bharti, Sanjay Kumar; Marosi, Krisztina; Froetscher, Lynn; Kassahun, Henok; Eckley, David Mark; Maul, Robert W; Bastian, Paul; De, Supriyo; Ghosh, Soumita; Nilsen, Hilde; Goldberg, Ilya G; Mattson, Mark P; Wilson, David M; Brosh, Robert M; Gorospe, Myriam; Bohr, Vilhelm A

2016-11-01

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

PubMed Central

2013-01-01

Background Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. PMID:24139482

Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Juanjuan; Zhang, Yu; Xu, Wentao, E-mail: xuwentaoboy@sina.com

Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did notmore » affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by OTA in vitro.« less

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

PubMed Central

Adeyemi, Richard O.; Landry, Sebastien; Davis, Meredith E.; Weitzman, Matthew D.; Pintel, David J.

2010-01-01

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells. PMID:20949077

Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koike, Manabu, E-mail: m_koike@nirs.go.jp; Yutoku, Yasutomo; Graduate School of Science, Chiba University, Chiba 263-8522

2011-08-19

Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 focimore » arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful tool as a detection marker of DNA damaged sites.« less

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

PubMed

Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

2016-09-01

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage

PubMed Central

Ciamporcero, Eric; Shen, He; Ramakrishnan, Swathi; Ku, Sheng Yu; Chintala, Sreenivasulu; Shen, Li; Adelaiye, Remi; Miles, Kiersten Marie; Ullio, Chiara; Pizzimenti, Stefania; Daga, Martina; Azabdaftari, Gissou; Attwood, Kris; Johnson, Candace; Zhang, Jianmin; Barrera, Giuseppina; Pili, Roberto

2015-01-01

Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knock-down sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC. PMID:26119935

[Stress-induced cellular adaptive mutagenesis].

PubMed

Zhu, Linjiang; Li, Qi

2014-04-01

The adaptive mutations exist widely in the evolution of cells, such as antibiotic resistance mutations of pathogenic bacteria, adaptive evolution of industrial strains, and cancerization of human somatic cells. However, how these adaptive mutations are generated is still controversial. Based on the mutational analysis models under the nonlethal selection conditions, stress-induced cellular adaptive mutagenesis is proposed as a new evolutionary viewpoint. The hypothetic pathway of stress-induced mutagenesis involves several intracellular physiological responses, including DNA damages caused by accumulation of intracellular toxic chemicals, limitation of DNA MMR (mismatch repair) activity, upregulation of general stress response and activation of SOS response. These responses directly affect the accuracy of DNA replication from a high-fidelity manner to an error-prone one. The state changes of cell physiology significantly increase intracellular mutation rate and recombination activity. In addition, gene transcription under stress condition increases the instability of genome in response to DNA damage, resulting in transcription-associated DNA mutagenesis. In this review, we summarize these two molecular mechanisms of stress-induced mutagenesis and transcription-associated DNA mutagenesis to help better understand the mechanisms of adaptive mutagenesis.

The Role of JMY in p53 Regulation.

PubMed

Adighibe, Omanma; Pezzella, Francesco

2018-05-31

Following the event of DNA damage, the level of tumour suppressor protein p53 increases inducing either cell cycle arrest or apoptosis. Junctional Mediating and Regulating Y protein (JMY) is a transcription co-factor involved in p53 regulation. In event of DNA damage, JMY levels also upregulate in the nucleus where JMY forms a co-activator complex with p300/CREB-binding protein (p300/CBP), Apoptosis-stimulating protein of p53 (ASPP) and Stress responsive activator of p53 (Strap). This co-activator complex then binds to and increases the ability of p53 to induce transcription of proteins triggering apoptosis but not cell cycle arrest. This then suggests that the increase of JMY levels due to DNA damage putatively "directs" p53 activity toward triggering apoptosis. JMY expression is also linked to increased cell motility as it: (1) downregulates the expression of adhesion molecules of the Cadherin family and (2) induces actin nucleation, making cells less adhesive and more mobile, favouring metastasis. All these characteristics taken together imply that JMY possesses both tumour suppressive and tumour metastasis promoting capabilities.

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

RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.

PubMed

Lam, Adeline R; Bert, Nina Le; Ho, Samantha Sw; Shen, Yu J; Tang, Li Fm; Xiong, Gordon M; Croxford, John L; Koo, Christine X; Ishii, Ken J; Akira, Shizuo; Raulet, David H; Gasser, Stephan

2014-04-15

The immunoreceptor NKG2D originally identified in natural killer (NK) cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DL) is induced by the DNA damage response (DDR), which is often activated constitutively in cancer cells, revealing them to NK cells as a mechanism of immunosurveillance. Here, we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines that express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. Irf3(+/-);Eμ-Myc mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared with Irf3(+/+);Eμ-Myc mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance. ©2014 AACR.

What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

PubMed Central

Brosey, Chris A.; Ahmed, Zamal; Lees-Miller, Susan P.; Tainer, John A.

2017-01-01

DNA damage outcomes depend upon the efficiency and fidelity of DNA damage responses (DDRs) for different cells and damage. As such, DDRs represent tightly regulated prototypical systems for linking nanoscale biomolecular structure and assembly to the biology of genomic regulation and cell signaling. However, the dynamic and multifunctional nature of DDR assemblies can render elusive the correlation between the structures of DDR factors and specific biological disruptions to the DDR when these structures are altered. In this chapter, we discuss concepts and strategies for combining structural, biophysical, and imaging techniques to investigate DDR recognition and regulation, and thus bridge sequence-level structural biochemistry to quantitative biological outcomes visualized in cells. We focus on representative DDR responses from PARP/PARG/AIF damage signaling in DNA single-strand break repair and nonhomologous end joining complexes in double-strand break repair. Methods with exemplary experimental results are considered with a focus on strategies for probing flexibility, conformational changes, and assembly processes that shape a predictive understanding of DDR mechanisms in a cellular context. Integration of structural and imaging measurements promises to provide foundational knowledge to rationally control and optimize DNA damage outcomes for synthetic lethality and for immune activation with resulting insights for biology and cancer interventions. PMID:28668129

Genotoxicity of Advanced Glycation End Products: Involvement of Oxidative Stress and of Angiotensin II Type 1 Receptors

NASA Astrophysics Data System (ADS)

Schupp, Nicole; Schinzel, Reinhard; Heidland, August; Stopper, Helga

2005-06-01

In patients with chronic renal failure, cancer incidence is increased. This may be related to an elevated level of genomic damage, which has been demonstrated by micronuclei formation as well as by comet assay analysis. Advanced glycation end products (AGEs) are markedly elevated in renal failure. In the comet assay, the model AGEs methylglyoxal- and carboxy(methyl)lysine-modified bovine serum albumin (BSA) induced significant DNA damage in colon, kidney, and liver cells. The addition of antioxidants prevented AGE-induced DNA damage, suggesting enhanced formation of reactive oxygen species (ROS). The coincubation with dimethylfumarate (DMF), an inhibitor of NF-κB translocation, reduced the genotoxic effect, thereby underscoring the key role of NF-κB in this process. One of the genes induced by NF-κB is angiotensinogen. The ensuing proteolytic activity yields angiotensin II, which evokes oxidative stress as well as proinflammatory responses. A modulator of the renin-angiotensin system (RAS), the angiotensin II (Ang II) receptor 1 antagonist, candesartan, yielded a reduction of the AGE-induced DNA damage, connecting the two signal pathways, RAS and AGE signaling. We were able to identify important participants in AGE-induced DNA damage: ROS, NF-κB, and Ang II, as well as modulators to prevent this DNA damage: antioxidants, DMF, and AT1 antagonists.

DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchetti, Francesco; Wyrobek, Andrew J.

Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization.more » During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.« less

Maintaining Genetic Integrity Under Extreme Conditions: Novel DNA Damage Repair Biology in the Archaea

DTIC Science & Technology

2013-11-23

Genetic analysis of Nre DNA repair function A4 Conclusions B. Widening the net in the search for new DNA-directed enzyme activities C. New tools for H...Figure 1) were hypothesised to be novel DNA repair enzymes . The stated aims of the proposal were to use a combination of genetic, biochemical and...in E.coli Almost all proteins that interact directly with PCNA are enzymes possessing DNA-directed activities such as nucleases, glycosylases

Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

2002-03-01

Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appearsmore » to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.« less

Evaluation of acetamiprid-induced genotoxic and oxidative responses in Eisenia fetida.

PubMed

Li, Bing; Xia, Xiaoming; Wang, Jinhua; Zhu, Lusheng; Wang, Jun; Wang, Guangchi

2018-06-19

As a novel neonicotinoids insecticide, acetamiprid has been widely used worldwide. In this study, a laboratory test was conducted to expose earthworms (Eisenia fetida) to artificial soil spiked with various concentrations of acetamiprid (0, 0.05, 0.10, 0.25 and 0.50 mg/kg of soil) respectively after 7, 14, 21 and 28 d. Reactive oxygen species (ROS) generation, antioxidant enzymes activity including superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferases (GST), malondialdehyde (MDA) content, and DNA damage were determined in earthworms. The ROS level increased in varying degrees at most exposure concentrations. The SOD activity was not significantly affected. The CAT activity was increased in the beginning, then gradually suppressed and resumed to the control level at the end, with the maximum change (171%) occurred at 14 d for 0.05 mg/kg. The GST activity was induced at 7 d, and then inhibited, with the maximum change (67.6%) occurred at 14 d for 0.50 mg/kg. The MDA content had a tendency that increasing at the first and decreasing at the end. The olive tail moment (OTM) in comet assay reflected a dose-dependent relationship, and DNA damage initially increased and then decreased over time. The results suggest that the sub-chronic exposure of acetamiprid can cause oxidative stress and DNA damage of earthworm and change the activity of the anti-oxidant enzyme. In addition, ROS content and DNA damage can be good indicators for assessing environmental risks of acetamiprid in earthworms. Copyright © 2018 Elsevier Inc. All rights reserved.

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers

PubMed Central

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-01-01

Objectives: This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. Methods: A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. Results: There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Conclusion: Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted. PMID:28077823

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.

Photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex.

PubMed

Zhang, Zhigang; Dai, Ruihui; Ma, Jiajia; Wang, Shuying; Wei, Xuehong; Wang, Hongfei

2015-02-01

Many planar photosensitizers tend to self-aggregate via van der Waals interactions between π-conjugated systems. The self-aggregation of the photosensitizer may reduce the efficiency of the photosensitizer to generate singlet oxygen, thereby diminishing its photodynamic activity. Efforts have been made to improve the photodynamic activity of bis-(o-diiminobenzosemiquinonato)platinum(II) which has planar geometry by the introduction of the sterically hindered triphenylamine moiety into the ligand. Herein we report the photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex in red light studied by fluorescence spectra, agarose gel assay and cell viability assay. The results suggest that the triphenylamine-modified platinum-diimine complex has better capability to generate singlet oxygen than bis-(o-diiminobenzosemiquinonato)platinum(II), and it can induce DNA damage in red light, causing high photocytotoxicity in HepG-2 cells in vitro. Copyright © 2014 Elsevier Inc. All rights reserved.

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

PubMed Central

Grabocka, Elda; Pylayeva-Gupta, Yuliya; Jones, Mathew JK; Lubkov, Veronica; Yemanaberhan, Eyoel; Taylor, Laura; Jeng, Hao Hsuan; Bar-Sagi, Dafna

2014-01-01

SUMMARY Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anti-cancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways, and consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo. PMID:24525237

Recruitment of TRF2 to laser-induced DNA damage sites.

PubMed

Huda, Nazmul; Abe, Satoshi; Gu, Ling; Mendonca, Marc S; Mohanty, Samarendra; Gilley, David

2012-09-01

Several lines of evidence suggest that the telomere-associated protein TRF2 plays critical roles in the DNA damage response. TRF2 is rapidly and transiently phosphorylated by an ATM-dependent pathway in response to DNA damage and this DNA damage-induced phosphoryation is essential for the DNA-PK-dependent pathway of DNA double-strand break repair (DSB). However, the type of DNA damage that induces TRF2 localization to the damage sites, the requirement for DNA damage-induced phosphorylation of TRF2 for its recruitment, as well as the detailed kinetics of TRF2 accumulation at DNA damage sites have not been fully investigated. In order to address these questions, we used an ultrafast femtosecond multiphoton laser and a continuous wave 405-nm single photon laser to induce DNA damage at defined nuclear locations. Our results showed that DNA damage produced by a femtosecond multiphoton laser was sufficient for localization of TRF2 to these DNA damage sites. We also demonstrate that ectopically expressed TRF2 was recruited to DNA lesions created by a 405-nm laser. Our data suggest that ATM and DNA-PKcs kinases are not required for TRF2 localization to DNA damage sites. Furthermore, we found that phosphorylation of TRF2 at residue T188 was not essential for its recruitment to laser-induced DNA damage sites. Thus, we provide further evidence that a protein known to function in telomere maintenance, TRF2, is recruited to sites of DNA damage and plays critical roles in the DNA damage response. Copyright © 2012 Elsevier Inc. All rights reserved.

Systemic lupus erythematosus in a multiethnic cohort (LUMINA): XXIX. Elevation of erythrocyte sedimentation rate is associated with disease activity and damage accrual.

PubMed

Vilá, Luis M; Alarcón, Graciela S; McGwin, Gerald; Bastian, Holly M; Fessler, Barri J; Reveille, John D

2005-11-01

To determine if different categories of erythrocyte sedimentation rate (ESR) elevation are associated with disease activity and/or damage in systemic lupus erythematosus (SLE). We studied 2317 study visits in 553 SLE patients (> or = 4 American College of Rheumatology criteria, < or = 5 years' disease duration at enrollment) from a multiethnic (Hispanic, African American, and Caucasian) longitudinal study of outcome. A study visit was done every 6 months for the first year and annually thereafter. Erythrocyte sedimentation rate (ESR) was measured using the Westergren method; results were expressed in 4 categories: < 25 (normal), 25-50 (mild elevation), 51-75 (moderate elevation), and > 75 (marked elevation) mm/h. Anti-dsDNA antibodies were measured at enrollment with the Crithidia luciliae assay. Disease activity was assessed with the Systemic Lupus Activity Measure (SLAM) and the Physician's Global Assessment (PGA). Because ESR is one of the measures evaluated in the SLAM, it was excluded from the total SLAM score. Disease damage was assessed with the Systemic Lupus International Collaborating Clinics damage index (SDI). The relationship between the SLAM (total and PGA) and SDI scores (at baseline and for all visits) and anti-dsDNA antibodies (at enrollment) with ESR was examined by univariable and generalized estimating equation (GEE) regression analyses. Ethnicity, age, and sex were entered in all regression models. The cohort consisted of 89.7% women with mean age 36.8 (SD 12.6) years and disease duration 4.6 (SD 3.2) years. GEE analyses showed that increasing levels of ESR and anti-dsDNA antibody positivity were independently associated with SLAM and PGA scores, at enrollment and for all visits. Overall, the associations of ESR with SLAM and PGA scores were stronger than for the presence of anti-dsDNA antibodies. At baseline, there was no relationship of ESR elevation or anti-dsDNA positivity with SDI scores. However, when all visits were studied, moderate and marked elevations of ESR were independently associated with SDI scores. Mild, moderate, and marked ESR elevations are strongly associated with disease activity in SLE. Moderate and marked ESR elevations are also associated with damage accrual. These associations are stronger than those for the presence of anti-dsDNA antibodies. Our data suggest that ESR could be used to assess disease activity and predict organ/system damage in a relatively rapid and inexpensive manner in SLE.

Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

PubMed Central

Benitez, Anaid; Yuan, Fenghua; Nakajima, Satoshi; Wei, Leizhen; Qian, Liangyue; Myers, Richard; Hu, Jennifer J.; Lan, Li; Zhang, Yanbin

2014-01-01

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner. PMID:24170812

Modulation of DNA-Induced Damage and Repair Capacity in Humans after Dietary Intervention with Lutein-Enriched Fermented Milk

PubMed Central

Herrero-Barbudo, Carmen; Soldevilla, Beatriz; Pérez-Sacristán, Belén; Blanco-Navarro, Inmaculada; Herrera, Mercedes; Granado-Lorencio, Fernando; Domínguez, Gemma

2013-01-01

Dietary factors provide protection against several forms of DNA damage. Additionally, consumer demand for natural products favours the development of bioactive food ingredients with health benefits. Lutein is a promising biologically active component in the food industry. The EFSA Panel on Dietetic Products, Nutrition and Allergies considers that protection from oxidative damage may be a beneficial physiological effect but that a cause and effect relationship has not been established. Thus, our aim was to evaluate the safety and potential functional effect of a lutein-enriched milk product using the Comet Assay in order to analyze the baseline, the induced DNA-damage and the repair capacity in the lymphocytes of 10 healthy donors before and after the intake of the mentioned product. Our data suggest that the regular consumption of lutein-enriched fermented milk results in a significant increase in serum lutein levels and this change is associated with an improvement in the resistance of DNA to damage and the capacity of DNA repair in lymphocytes. Our results also support the lack of a genotoxic effect at the doses supplied as well as the absence of interactions and side effects on other nutritional and biochemicals markers. PMID:24040187

DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea

PubMed Central

Jones, Daniel L.; Baxter, Bonnie K.

2017-01-01

Halophilic archaea push the limits of life at several extremes. In particular, they are noted for their biochemical strategies in dealing with osmotic stress, low water activity and cycles of desiccation in their hypersaline environments. Another feature common to their habitats is intense ultraviolet (UV) radiation, which is a challenge that microorganisms must overcome. The consequences of high UV exposure include DNA lesions arising directly from bond rearrangement of adjacent bipyrimidines, or indirectly from oxidative damage, which may ultimately result in mutation and cell death. As such, these microorganisms have evolved a number of strategies to navigate the threat of DNA damage, which we differentiate into two categories: DNA repair and photoprotection. Photoprotection encompasses damage avoidance strategies that serve as a “first line of defense,” and in halophilic archaea include pigmentation by carotenoids, mechanisms of oxidative damage avoidance, polyploidy, and genomic signatures that make DNA less susceptible to photodamage. Photolesions that do arise are addressed by a number of DNA repair mechanisms that halophilic archaea efficiently utilize, which include photoreactivation, nucleotide excision repair, base excision repair, and homologous recombination. This review seeks to place DNA damage, repair, and photoprotection in the context of halophilic archaea and the solar radiation of their hypersaline environments. We also provide new insight into the breadth of strategies and how they may work together to produce remarkable UV-resistance for these microorganisms. PMID:29033920

Xeroderma Pigmentosum Group C Deficiency Alters Cigarette Smoke DNA Damage Cell Fate and Accelerates Emphysema Development.

PubMed

Sears, Catherine R; Zhou, Huaxin; Justice, Matthew J; Fisher, Amanda J; Saliba, Jacob; Lamb, Isaac; Wicker, Jessica; Schweitzer, Kelly S; Petrache, Irina

2018-03-01

Cigarette smoke (CS) exposure is a major risk factor for the development of emphysema, a common disease characterized by loss of cells comprising the lung parenchyma. The mechanisms of cell injury leading to emphysema are not completely understood but are thought to involve persistent cytotoxic or mutagenic DNA damage induced by CS. Using complementary cell culture and mouse models of CS exposure, we investigated the role of the DNA repair protein, xeroderma pigmentosum group C (XPC), on CS-induced DNA damage repair and emphysema. Expression of XPC was decreased in mouse lungs after chronic CS exposure and XPC knockdown in cultured human lung epithelial cells decreased their survival after CS exposure due to activation of the intrinsic apoptosis pathway. Similarly, cell autophagy and apoptosis were increased in XPC-deficient mouse lungs and were further increased by CS exposure. XPC deficiency was associated with structural and functional changes characteristic of emphysema, which were worsened by age, similar to levels observed with chronic CS exposure. Taken together, these findings suggest that repair of DNA damage by XPC plays an important and previously unrecognized role in the maintenance of alveolar structures. These findings support that loss of XPC, possibly due to chronic CS exposure, promotes emphysema development and further supports a link between DNA damage, impaired DNA repair, and development of emphysema.

DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea.

PubMed

Jones, Daniel L; Baxter, Bonnie K

2017-01-01

Halophilic archaea push the limits of life at several extremes. In particular, they are noted for their biochemical strategies in dealing with osmotic stress, low water activity and cycles of desiccation in their hypersaline environments. Another feature common to their habitats is intense ultraviolet (UV) radiation, which is a challenge that microorganisms must overcome. The consequences of high UV exposure include DNA lesions arising directly from bond rearrangement of adjacent bipyrimidines, or indirectly from oxidative damage, which may ultimately result in mutation and cell death. As such, these microorganisms have evolved a number of strategies to navigate the threat of DNA damage, which we differentiate into two categories: DNA repair and photoprotection. Photoprotection encompasses damage avoidance strategies that serve as a "first line of defense," and in halophilic archaea include pigmentation by carotenoids, mechanisms of oxidative damage avoidance, polyploidy, and genomic signatures that make DNA less susceptible to photodamage. Photolesions that do arise are addressed by a number of DNA repair mechanisms that halophilic archaea efficiently utilize, which include photoreactivation, nucleotide excision repair, base excision repair, and homologous recombination. This review seeks to place DNA damage, repair, and photoprotection in the context of halophilic archaea and the solar radiation of their hypersaline environments. We also provide new insight into the breadth of strategies and how they may work together to produce remarkable UV-resistance for these microorganisms.

Effects of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites on DNA damage and repair under in vitro conditions.

PubMed

Ozcagli, Eren; Alpertunga, Buket; Fenga, Concettina; Berktas, Mehmet; Tsitsimpikou, Christina; Wilks, Martin F; Tsatsakis, Αristidis M

2016-03-01

3-monochloropropane-1,2-diol (3-MCPD) is a food contaminant that occurs during industrial production processes and can be found mainly in fat and salt containing products. 3-MCPD has exhibited mutagenic activity in vitro but not in vivo, however, a genotoxic mechanism for the occurrence of kidney tumors has not so far been excluded. The main pathway of mammalian 3-MCPD metabolism is via the formation of β--chlorolactatic acid and formation of glycidol has been demonstrated in bacterial metabolism. The aim of this study was to investigate genotoxic and oxidative DNA damaging effects of 3-MCPD and its metabolites, and to provide a better understanding of their roles in DNA repair processes. DNA damage was assessed by alkaline comet assay in target rat kidney epithelial cell lines (NRK-52E) and human embryonic kidney cells (HEK-293). Purine and pyrimidine base damage, H2O2 sensitivity and DNA repair capacity were assessed via modified comet assay. The results revealed in vitro evidence for increased genotoxicity and H2O2 sensitivity. No association was found between oxidative DNA damage and DNA repair capacity with the exception of glycidol treatment at 20 μg/mL. These findings provide further insights into the mechanisms underlying the in vitro genotoxic potential of 3-MCPD and metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

Grapefruit juice intake does not enhance but rather protects against aflatoxin B1-induced liver DNA damage through a reduction in hepatic CYP3A activity.

PubMed

Miyata, Masaaki; Takano, Hiroki; Guo, Lian Q; Nagata, Kiyoshi; Yamazoe, Yasushi

2004-02-01

Influence of grapefruit juice intake on aflatoxin B1 (AFB1)-induced liver DNA damage was examined using a Comet assay in F344 rats given 5 mg/kg AFB1 by gavage. Rats allowed free access to grapefruit juice for 5 days prior to AFB1 administration resulted in clearly reduced DNA damage in liver, to 65% of the level in rats that did not receive grapefruit juice. Furthermore, rats treated with grapefruit juice extract (100 mg/kg per os) for 5 days prior to AFB1 treatment also reduced the DNA damage to 74% of the level in rats that did not receive grapefruit juice. No significant differences in the portal blood and liver concentrations of AFB1 were observed between grapefruit juice intake rats and the controls. In an Ames assay with AFB1 using Salmonella typhimurium TA98, lower numbers of revertant colonies were detected with hepatic microsomes prepared from rats administered grapefruit juice, compared with those from control rats. Microsomal testosterone 6beta-hydroxylation was also lower with rats given grapefruit juice than with control rats. Immunoblot analyses showed a significant decrease in hepatic CYP3A content, but not CYP1A and CYP2C content, in microsomes of grapefruit juice-treated rats than in non-treated rats. No significant difference in hepatic glutathione S-transferase (GST) activity and glutathione content was observed in the two groups. GSTA5 protein was not detected in hepatic cytosol of the two groups. In microsomal systems, grapefruit juice extract inhibited AFB1-induced mutagenesis in the presence of a microsomal activation system from livers of humans as well as rats. These results suggest that grapefruit juice intake suppresses AFB1-induced liver DNA damage through inactivation of the metabolic activation potency for AFB1 in rat liver.

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

PubMed

Haendeler, Judith; Dröse, Stefan; Büchner, Nicole; Jakob, Sascha; Altschmied, Joachim; Goy, Christine; Spyridopoulos, Ioakim; Zeiher, Andreas M; Brandt, Ulrich; Dimmeler, Stefanie

2009-06-01

The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

Myricitrin and bioactive extract of Albizia amara leaves: DNA protection and modulation of fertility and antioxidant-related genes expression.

PubMed

Kassem, Mona El Said; Ibrahim, Lamya Fawzy; Hussein, Sameh Reda; El-Sharawy, Reham; El-Ansari, Mohamed Amin; Hassanane, Mahrousa Mohamed; Booles, Hoda Fahime

2016-11-01

Albizia species are reported to exhibit many biological activities including antiovulatory properties in female rats and antispermatogenic and antiandrogenic activities in male rats. The present study investigates the flavonoids of Albizia amara (Roxb.) B. Boivin (Fabaceae) leaves and evaluates their activity on gene expression of fertility and antioxidant glutathione-S-transferase-related genes of treated female mice in addition to their effect on DNA damage. Plant materials were extracted by using 70% methanol for 48 h, the extract was chromatographed on a polyamide 6S column, each isolated compound was purified by using Sephadex LH-20 column; its structure was elucidated by chemical and spectral methods. Both the leaves extract and myricitrin (200, 30 mg/kg bw/d, respectively) were assayed for their effect on DNA damage in female mice after four weeks treatment using Comet assay. Their modulatory activity on gene expression of fertility (aromatase CYP19 and luteinizing hormone LH) and antioxidant glutathione-S-transferase (GST)-related genes of treated female mice were investigated by real-time PCR (qPCR). Quercetin-3-O-gentiobioside, myricitrin, quercetin-3-O-α-rhamnopyranoside, myricetin, quercetin and kaempferol were isolated and identified from the studied taxa. Myricitrin and the extract induced low rate of DNA damage (4.8% and 5%, respectively), compared with the untreated control (4.2%) and significantly down-regulated the expression of CYP19 and LH genes and up-regulated GST gene. Our results highlight the potential effect of the leaves extract of Albizia amara and myricitrin as fertility-regulating phytoconstituents with ability to protect DNA from damage and cells from oxidative stress.

Transcription-Coupled Repair and Complex Biology.

PubMed

Portman, James R; Strick, Terence R

2018-05-04

All active living organisms mitigate DNA damage via DNA repair, and the so-called nucleotide excision repair pathway (NER) represents a functionally major part of the cell's DNA repair repertoire [1]. In this pathway, the damaged strand of DNA is incised and removed before being resynthesized. This form of DNA repair requires a multitude of proteins working in a complex choreography. Repair thus typically involves detection of a DNA lesion; validation of that detection event; search for an appropriate incision site and subsequent DNA incision; DNA unwinding/removal; and DNA resynthesis and religation. These activities are ultimately the result of molecules randomly diffusing and bumping into each other and acting in succession. It is also true however that repair components are often assembled into functional complexes which may be more efficient or regular in their mode of action. Studying DNA repair complexes for their mechanisms of assembly, action, and disassembly can help address fundamental questions such as whether DNA repair pathways are branched or linear; whether for instance they tolerate fluctuations in numbers of components; and more broadly how search processes between macromolecules take place or can be enhanced. Copyright © 2018. Published by Elsevier Ltd.

Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.

2013-12-22

Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing amore » ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.« less

Loss of DNA mismatch repair imparts a selective advantage in planarian adult stem cells.

PubMed

Hollenbach, Jessica P; Resch, Alissa M; Palakodeti, Dasaradhi; Graveley, Brenton R; Heinen, Christopher D

2011-01-01

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis.

Loss of DNA Mismatch Repair Imparts a Selective Advantage in Planarian Adult Stem Cells

PubMed Central

Hollenbach, Jessica P.; Resch, Alissa M.; Palakodeti, Dasaradhi; Graveley, Brenton R.; Heinen, Christopher D.

2011-01-01

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis. PMID:21747960

Molecular and sensory mechanisms to mitigate sunlight-induced DNA damage in treefrog tadpoles.

PubMed

Schuch, André P; Lipinski, Victor M; Santos, Mauricio B; Santos, Caroline P; Jardim, Sinara S; Cechin, Sonia Z; Loreto, Elgion L S

2015-10-01

The increased incidence of solar ultraviolet B (UVB) radiation has been proposed as an environmental stressor, which may help to explain the enigmatic decline of amphibian populations worldwide. Despite growing knowledge regarding the UV-induced biological effects in several amphibian models, little is known about the efficacy of DNA repair pathways. In addition, little attention has been given to the interplay between these molecular mechanisms with other physiological strategies that avoid the damage induced by sunlight. Here, DNA lesions induced by environmental doses of solar UVB and UVA radiation were detected in genomic DNA samples of treefrog tadpoles (Hypsiboas pulchellus) and their DNA repair activity was evaluated. These data were complemented by monitoring the induction of apoptosis in blood cells and tadpole survival. Furthermore, the tadpoles' ability to perceive and escape from UV wavelengths was evaluated as an additional strategy of photoprotection. The results show that tadpoles are very sensitive to UVB light, which could be explained by the slow DNA repair rates for both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6,4) pyrimidone photoproducts (6,4PPs). However, they were resistant to UVA, probably as a result of the activation of photolyases during UVA irradiation. Surprisingly, a sensory mechanism that triggers their escape from UVB and UVA light avoids the generation of DNA damage and helps to maintain the genomic integrity. This work demonstrates the genotoxic impact of both UVB and UVA radiation on tadpoles and emphasizes the importance of the interplay between molecular and sensory mechanisms to minimize the damage caused by sunlight. © 2015. Published by The Company of Biologists Ltd.

Phosphorylated (pT371)TRF1 is recruited to sites of DNA damage to facilitate homologous recombination and checkpoint activation

PubMed Central

McKerlie, Megan; Walker, John R.; Mitchell, Taylor R. H.; Wilson, Florence R.; Zhu, Xu-Dong

2013-01-01

TRF1, a duplex telomeric DNA-binding protein, plays an important role in telomere metabolism. We have previously reported that a fraction of endogenous TRF1 can stably exist free of telomere chromatin when it is phosphorylated at T371 by Cdk1; however, the role of this telomere-free (pT371)TRF1 has yet to be fully characterized. Here we show that phosphorylated (pT371)TRF1 is recruited to sites of DNA damage, forming damage-induced foci in response to ionizing radiation (IR), etoposide and camptothecin. We find that IR-induced (pT371)TRF1 foci formation is dependent on the ATM- and Mre11/Rad50/Nbs1-mediated DNA damage response. While loss of functional BRCA1 impairs the formation of IR-induced (pT371)TRF1 foci, depletion of either 53BP1 or Rif1 stimulates IR-induced (pT371)TRF1 foci formation. In addition, we show that TRF1 depletion or the lack of its phosphorylation at T371 impairs DNA end resection and repair of nontelomeric DNA double-strand breaks by homologous recombination. The lack of TRF1 phosphorylation at T371 also hampers the activation of the G2/M checkpoint and sensitizes cells to PARP inhibition, IR and camptothecin. Collectively, these results reveal a novel but important function of phosphorylated (pT371)TRF1 in facilitating DNA double-strand break repair and the maintenance of genome integrity. PMID:23997120

Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet

PubMed Central

Hsiao, Pi-Jung; Hsieh, Tusty-Jiuan; Kuo, Kung-Kai; Hung, Wei-Wen; Tsai, Kun-Bow; Yang, Ching-Hsiu; Yu, Ming-Lung; Shin, Shyi-Jang

2008-01-01

Background Pioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2) and DNA glycosylase (Ogg1, MutY). Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression. Results Our results show that hepatic steatosis was induced by a high-fat diet and improved by adding pioglitazone. Malondialdehyde concentration and 8-oxoG staining were strongly increased in the high-fat diet group, but attenuated by pioglitazone. Gene expressions of antioxidant defense mechanism: Sod1, Sod2, Ogg1 and MutY significantly decreased in the high-fat diet group but reversed by pioglitazone co-administration. Conclusion The attenuation of hepatic oxidative DNA damage by pioglitazone in a high-fat diet may be mediated by up-regulation of the antioxidant defense mechanism and oxidative DNA repair activity. The diminution of oxidative damage may explain the clinical benefit of pioglitazone treatment in patients with non-alcoholic fatty liver disease. PMID:18822121

Review of Chromium (VI) Apoptosis, Cell-Cycle-Arrest, and Carcinogenesis

PubMed Central

Chiu, A; Shi, J; Lee, WKP; Hill, R; Wakeman, TP; Katz, A; Xu, B; Dalal, NS; Robertson, JD; Chen, C; Chiu, N; Donehower, L

2014-01-01

Hexavalent chromium combines with glutathione in chloride intracellular channel carrier to form tetravalent and pentavelent chromium in plasma and organelle membranes. It also combines with NADH/NADPH to form pentavalent chromium in mitochondria. Tetravalent- and pentavalent- chromium (directly and indirectly) mediated DNA double strand breaks activate DNA damage signaling sensors: DNA-dependent-protein-kinase signals p53-dependent intrinsic mitochorndrial apoptosis, and ataxia-telangiectasia-mutated and ataxia-telangiectasia-Rad3-related signal cell-arrest for DNA repair. Tetravalent chromium may be the most potent species since it causes DNA breaks and somatic recombination, but not apoptosis. Upon further failure of apoptosis and senescence/DNA-repair, damaged cells may become immortal with loss-of-heterozygosity and genetic plasticity. PMID:20859824

Host DNA repair proteins in response to Pseudomonas aeruginosa in lung epitehlial cells and in mice

USDA-ARS?s Scientific Manuscript database

Host DNA damage and DNA repair response to bacterial infections and its significance are not fully understood. Here, we demonstrate that infection by Gram-negative bacterium P. aeruginosa significantly altered the expression and enzymatic activity of base excision DNA repair protein OGG1 in lung epi...

Sulfur and selenium antioxidants: challenging radical scavenging mechanisms and developing structure-activity relationships based on metal binding.

PubMed

Zimmerman, Matthew T; Bayse, Craig A; Ramoutar, Ria R; Brumaghim, Julia L

2015-04-01

Because sulfur and selenium antioxidants can prevent oxidative damage, numerous animal and clinical trials have investigated the ability of these compounds to prevent the oxidative stress that is an underlying cause of cardiovascular disease, Alzheimer's disease, and cancer, among others. One of the most common sources of oxidative damage is metal-generated hydroxyl radical; however, very little research has focused on determining the metal-binding abilities and structural attributes that affect oxidative damage prevention by sulfur and selenium compounds. In this review, we describe our ongoing investigations into sulfur and selenium antioxidant prevention of iron- and copper-mediated oxidative DNA damage. We determined that many sulfur and selenium compounds inhibit Cu(I)-mediated DNA damage and that DNA damage prevention varies dramatically when Fe(II) is used in place of Cu(I) to generate hydroxyl radical. Oxidation potentials of the sulfur or selenium compounds do not correlate with their ability to prevent DNA damage, highlighting the importance of metal coordination rather than reactive oxygen species scavenging as an antioxidant mechanism. Additional gel electrophoresis, mass spectrometry, and UV-visible studies confirmed sulfur and selenium antioxidant binding to Cu(I) and Fe(II). Ultimately, our studies established that both the hydroxyl-radical-generating metal ion and the chemical environment of the sulfur or selenium significantly affect DNA damage prevention and that metal coordination is an essential mechanism for these antioxidants. Copyright © 2015 Elsevier Inc. All rights reserved.