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Sample records for affect nonhomologous end-joining

  1. The endless tale of non-homologous end-joining.

    Science.gov (United States)

    Weterings, Eric; Chen, David J

    2008-01-01

    DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.

  2. The endless tale of non-homologous end-joining

    Institute of Scientific and Technical Information of China (English)

    Eric Weterings; David J Chen

    2008-01-01

    DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addi-tion, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.

  3. The minimal Bacillus subtilis nonhomologous end joining repair machinery.

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    Miguel de Vega

    Full Text Available It is widely accepted that repair of double-strand breaks in bacteria that either sporulate or that undergo extended periods of stationary phase relies not only on homologous recombination but also on a minimal nonhomologous end joining (NHEJ system consisting of a dedicated multifunctional ATP-dependent DNA Ligase D (LigD and the DNA-end-binding protein Ku. Bacillus subtilis is one of the bacterial members with a NHEJ system that contributes to genome stability during the stationary phase and germination of spores, having been characterized exclusively in vivo. Here, the in vitro analysis of the functional properties of the purified B. subtilis LigD (BsuLigD and Ku (BsuKu proteins is presented. The results show that the essential biochemical signatures exhibited by BsuLigD agree with its proposed function in NHEJ: i inherent polymerization activity showing preferential insertion of NMPs, ii specific recognition of the phosphate group at the downstream 5' end, iii intrinsic ligase activity, iv ability to promote realignments of the template and primer strands during elongation of mispaired 3' ends, and v it is recruited to DNA by BsuKu that stimulates the inherent polymerization and ligase activities of the enzyme allowing it to deal with and to hold different and unstable DNA realignments.

  4. Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency.

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    Bhargava, Ragini; Carson, Caree R; Lee, Gabriella; Stark, Jeremy M

    2017-01-24

    A likely mechanism of chromosomal rearrangement formation involves joining the ends from two different chromosomal double-strand breaks (DSBs). These events could potentially be mediated by either of two end-joining (EJ) repair pathways [canonical nonhomologous end joining (C-NHEJ) or alternative end joining (ALT-EJ)], which cause distinct rearrangement junction patterns. The relative role of these EJ pathways during rearrangement formation has remained controversial. Along these lines, we have tested whether the DNA damage response mediated by the Ataxia Telangiectasia Mutated (ATM) kinase may affect the relative influence of C-NHEJ vs. ALT-EJ on rearrangement formation. We developed a reporter in mouse cells for a 0.4-Mbp deletion rearrangement that is formed by EJ between two DSBs induced by the Cas9 endonuclease. We found that disruption of the ATM kinase causes an increase in the frequency of the rearrangement as well as a shift toward rearrangement junctions that show hallmarks of C-NHEJ. Furthermore, ATM suppresses rearrangement formation in an experimental condition, in which C-NHEJ is the predominant EJ repair event (i.e., expression of the 3' exonuclease Trex2). Finally, several C-NHEJ factors are required for the increase in rearrangement frequency caused by inhibition of the ATM kinase. We also examined ATM effectors and found that H2AX shows a similar influence as ATM, whereas the influence of ATM on this rearrangement seems independent of 53BP1. We suggest that the contribution of the C-NHEJ pathway to the formation of a 0.4-Mbp deletion rearrangement is enhanced in ATM-deficient cells.

  5. The role of RecQ helicases in non-homologous end-joining

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    Keijzers, Guido; Maynard, Scott; Shamanna, Raghavendra A;

    2014-01-01

    Abstract DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double......-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA...... termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V...

  6. Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells*

    OpenAIRE

    Liu, Jia; Majumdar, Alokes; Liu, Jilan; Thompson, Lawrence H.; Seidman, Michael M.

    2010-01-01

    Double strand breaks (DSBs) can be repaired by homology independent nonhomologous end joining (NHEJ) pathways involving proteins such as Ku70/80, DNAPKcs, Xrcc4/Ligase 4, and the Mre11/Rad50/Nbs1 (MRN) complex. DSBs can also be repaired by homology-dependent pathways (HDR), in which the MRN and CtIP nucleases produce single strand ends that engage homologous sequences either by strand invasion or strand annealing. The entry of ends into HDR pathways underlies protocols for genomic manipulatio...

  7. The Ku70/80 ring in Non-Homologous End-Joining

    DEFF Research Database (Denmark)

    Kragelund, Birthe Brandt; Weterings, Eric; Hartmann-Petersen, Rasmus;

    2016-01-01

    Non-homologous end-joining (NHEJ) is an essential DNA double strand break repair pathway during all cell cycle stages. Deficiency in NHEJ factors can lead to accumulation of unrepaired DNA breaks or faulty DNA repair, which may ultimately result in cell death, senescence or carcinogenesis. The Ku70....../80 heterodimer is a key-player in the NHEJ pathway and binds to DNA termini with high affinity, where it helps to protect DNA ends from degradation and to recruit other NHEJ factors required for repair. The mechanism of Ku70/80 detachment from the DNA helix after completion of DNA repair is incompletely...

  8. Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

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    Cynthia L. Hendrickson

    2010-01-01

    Full Text Available In mammalian cells, DNA double-strand breaks (DSBs are primarily repaired by nonhomologous end joining (NHEJ. The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcs to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PKcs appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA-PKcs protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.

  9. Positive selection on the nonhomologous end-joining factor Cernunnos-XLF in the human lineage

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    Jurka Jerzy

    2006-06-01

    Full Text Available Abstract Background Cernunnos-XLF is a nonhomologous end-joining factor that is mutated in patients with a rare immunodeficiency with microcephaly. Several other microcephaly-associated genes such as ASPM and microcephalin experienced recent adaptive evolution apparently linked to brain size expansion in humans. In this study we investigated whether Cernunnos-XLF experienced similar positive selection during human evolution. Results We obtained or reconstructed full-length coding sequences of chimpanzee, rhesus macaque, canine, and bovine Cernunnos-XLF orthologs from sequence databases and sequence trace archives. Comparison of coding sequences revealed an excess of nonsynonymous substitutions consistent with positive selection on Cernunnos-XLF in the human lineage. The hotspots of adaptive evolution are concentrated around a specific structural domain, whose analogue in the structurally similar XRCC4 protein is involved in binding of another nonhomologous end-joining factor, DNA ligase IV. Conclusion Cernunnos-XLF is a microcephaly-associated locus newly identified to be under adaptive evolution in humans, and possibly played a role in human brain expansion. We speculate that Cernunnos-XLF may have contributed to the increased number of brain cells in humans by efficient double strand break repair, which helps to prevent frequent apoptosis of neuronal progenitors and aids mitotic cell cycle progression. Reviewers This article was reviewed by Chris Ponting and Richard Emes (nominated by Chris Ponting, Kateryna Makova, Gáspár Jékely and Eugene V. Koonin.

  10. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

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    Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: tkkohno@ncc.go.jp [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  11. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

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    Simsek, Deniz; Brunet, Erika; Wong, Sunnie Yan-Wai; Katyal, Sachin; Gao, Yankun; McKinnon, Peter J; Lou, Jacqueline; Zhang, Lei; Li, James; Rebar, Edward J; Gregory, Philip D; Holmes, Michael C; Jasin, Maria

    2011-06-01

    Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

  12. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

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    Deniz Simsek

    2011-06-01

    Full Text Available Nonhomologous end-joining (NHEJ is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4, suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

  13. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining.

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    Richard Bowater

    2006-02-01

    Full Text Available DNA double-strand breaks (DSBs are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ. Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.

  14. KAP1 Deacetylation by SIRT1 Promotes Non-Homologous End-Joining Repair.

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    Yi-Hui Lin

    Full Text Available Homologous recombination and non-homologous end joining are two major DNA double-strand-break repair pathways. While HR-mediated repair requires a homologous sequence as the guiding template to restore the damage site precisely, NHEJ-mediated repair ligates the DNA lesion directly and increases the risk of losing nucleotides. Therefore, how a cell regulates the balance between HR and NHEJ has become an important issue for maintaining genomic integrity over time. Here we report that SIRT1-dependent KAP1 deacetylation positively regulates NHEJ. We show that up-regulation of KAP1 attenuates HR efficiency while promoting NHEJ repair. Moreover, SIRT1-mediated KAP1 deacetylation further enhances the effect of NHEJ by stabilizing its interaction with 53BP1, which leads to increased 53BP1 focus formation in response to DNA damage. Taken together, our study suggests a SIRT1-KAP1 regulatory mechanism for HR-NHEJ repair pathway choice.

  15. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.

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    Ghezraoui, Hind; Piganeau, Marion; Renouf, Benjamin; Renaud, Jean-Baptiste; Sallmyr, Annahita; Ruis, Brian; Oh, Sehyun; Tomkinson, Alan E; Hendrickson, Eric A; Giovannangeli, Carine; Jasin, Maria; Brunet, Erika

    2014-09-18

    Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

  16. ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma.

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    Koschmann, Carl; Calinescu, Anda-Alexandra; Nunez, Felipe J; Mackay, Alan; Fazal-Salom, Janet; Thomas, Daniel; Mendez, Flor; Kamran, Neha; Dzaman, Marta; Mulpuri, Lakshman; Krasinkiewicz, Johnathon; Doherty, Robert; Lemons, Rosemary; Brosnan-Cashman, Jacqueline A; Li, Youping; Roh, Soyeon; Zhao, Lili; Appelman, Henry; Ferguson, David; Gorbunova, Vera; Meeker, Alan; Jones, Chris; Lowenstein, Pedro R; Castro, Maria G

    2016-03-02

    Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

  17. Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

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    Rossner, Pavel, E-mail: prossner@biomed.cas.cz; Rossnerova, Andrea; Beskid, Olena; Tabashidze, Nana; Libalova, Helena; Uhlirova, Katerina; Topinka, Jan; Sram, Radim J.

    2014-05-15

    Highlights: • We analyzed the effect of air pollutants on NHEJ and chromosome aberrations. • In HEL12469 cells B[a]P and extractable organic matter induced DSBs. • The compounds induced XRCC4 expression and a weak Ku70/80 response. • We found increased frequency of aberrations of chromosomes 1, 2, 4, 5, 7 and 17. • The tested compounds preferentially affected chromosome 7. - Abstract: In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24 h with the following concentrations of tested chemicals: B[a]P: 1 μM, 10 μM, 25 μM; EOMs: 1 μg/ml, 10 μg/ml, 25 μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25 μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and

  18. ATM limits incorrect end utilization during non-homologous end joining of multiple chromosome breaks.

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    Bennardo, Nicole; Stark, Jeremy M

    2010-11-04

    Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ) repair of multiple chromosomal double-strand breaks (DSBs). We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ) or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs). In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI-resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ) factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ.

  19. ATM limits incorrect end utilization during non-homologous end joining of multiple chromosome breaks.

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    Nicole Bennardo

    2010-11-01

    Full Text Available Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ repair of multiple chromosomal double-strand breaks (DSBs. We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs. In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI-resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ.

  20. Transient disruption of non-homologous end-joining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans

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    Nielsen, Jakob Blæsbjerg; Nielsen, Michael Lynge; Mortensen, Uffe Hasbro

    2008-01-01

    We have developed a transiently disrupted nkuA system in Aspergillus nidulans for efficient gene targeting. The nkuA disruption was made by inserting a counter-selectable marker flanked by a direct repeat (DR) composed of nkuA sequences. In the disrupted state, the non-homologous end-joining (NHE...

  1. Comparison of nonhomologous end joining and homologous recombination in human cells

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    Mao, Zhiyong; Bozzella, Michael; Seluanov, Andrei; Gorbunova, Vera

    2009-01-01

    The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR leads to accurate repair, while NHEJ is intrinsically mutagenic. To understand human somatic mutation it is essential to know the relationship between these pathways in human cells. Here we provide a comparison of the kinetics and relative contributions of HR and NHEJ in normal human cells. We used chromosomally integrated fluorescent reporter substrates for real-time in vivo monitoring of the NHEJ and HR. By examining multiple integrated clones we show that the efficiency of NHEJ and HR is strongly influenced by chromosomal location. Furthermore, we show that NHEJ of compatible ends (NHEJ-C) and NHEJ of incompatible ends (NHEJ-I) are fast processes, which can be completed in approximately 30 min, while HR is much slower and takes 7h or longer to complete. In actively cycling cells NHEJ-C is twice as efficient as NHEJ-I, and NHEJ-I is three times more efficient than HR. Our results suggest that NHEJ is a faster and more efficient DSB repair pathway than HR. PMID:18675941

  2. Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining

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    Rebecca Cook

    2015-03-01

    Full Text Available Deficiencies in DNA double-strand break (DSB repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1 is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ. Support of cNHEJ involves a mechanism independent of RB1’s cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution.

  3. Sequence conversion by single strand oligonucleotide donors via non-homologous end joining in mammalian cells.

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    Liu, Jia; Majumdar, Alokes; Liu, Jilan; Thompson, Lawrence H; Seidman, Michael M

    2010-07-23

    Double strand breaks (DSBs) can be repaired by homology independent nonhomologous end joining (NHEJ) pathways involving proteins such as Ku70/80, DNAPKcs, Xrcc4/Ligase 4, and the Mre11/Rad50/Nbs1 (MRN) complex. DSBs can also be repaired by homology-dependent pathways (HDR), in which the MRN and CtIP nucleases produce single strand ends that engage homologous sequences either by strand invasion or strand annealing. The entry of ends into HDR pathways underlies protocols for genomic manipulation that combine site-specific DSBs with appropriate informational donors. Most strategies utilize long duplex donors that participate by strand invasion. Work in yeast indicates that single strand oligonucleotide (SSO) donors are also active, over considerable distance, via a single strand annealing pathway. We examined the activity of SSO donors in mammalian cells at DSBs induced either by a restriction nuclease or by a targeted interstrand cross-link. SSO donors were effective immediately adjacent to the break, but activity declined sharply beyond approximately 100 nucleotides. Overexpression of the resection nuclease CtIP increased the frequency of SSO-mediated sequence modulation distal to the break site, but had no effect on the activity of an SSO donor adjacent to the break. Genetic and in vivo competition experiments showed that sequence conversion by SSOs in the immediate vicinity of the break was not by strand invasion or strand annealing pathways. Instead these donors competed for ends that would have otherwise entered NHEJ pathways.

  4. Structural Basis of DNA Ligase IV-Artemis Interaction in Nonhomologous End-Joining

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    Pablo De Ioannes

    2012-12-01

    Full Text Available DNA ligase IV (LigIV and Artemis are central components of the nonhomologous end-joining (NHEJ machinery that is required for V(DJ recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ.

  5. Structural Biology of DNA Repair: Spatial Organisation of the Multicomponent Complexes of Nonhomologous End Joining

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    Takashi Ochi

    2010-01-01

    Full Text Available Nonhomologous end joining (NHEJ plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods—X-ray crystallography, electron microscopy and small angle X-ray scattering—can give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higher-order complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere.

  6. DNA ligases I and III cooperate in alternative non-homologous end-joining in vertebrates.

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    Katja Paul

    Full Text Available Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs from their genomes predominantly by two non-homologous end joining (NHEJ pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3.

  7. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells

    Science.gov (United States)

    Mao, Zhiyong; Bozzella, Michael; Seluanov, Andrei; Gorbunova, Vera

    2009-01-01

    DNA double-strand breaks (DSBs) are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an intrinsically error-prone pathway while HR results in accurate repair. To understand the origin of genomic instability in human cells it is important to know the contribution of each DSB repair pathway. Studies of rodent cells and human cancer cell lines have shown that the choice between NHEJ or HR pathways depends on cell cycle stage. Surprisingly, cell cycle regulation of DSB repair has not been examined in normal human cells with intact cell cycle checkpoints. Here we measured the efficiency of NHEJ and HR at different cell cycle stages in hTERT-immortalized diploid human fibroblasts. We utilized cells with chromosomally-integrated fluorescent reporter cassettes, in which a unique DSB is introduced by a rare-cutting endonuclease. We show that NHEJ is active throughout the cell cycle, and its activity increases as cells progress from G1 to G2/M (G1

  8. Modeling damage complexity-dependent non-homologous end-joining repair pathway.

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    Yongfeng Li

    Full Text Available Non-homologous end joining (NHEJ is the dominant DNA double strand break (DSB repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed.

  9. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

    Science.gov (United States)

    Arras, Samantha D M; Fraser, James A

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well.

  10. RECQ HELICASE RECQL4 PARTICIPATES IN NON-HOMOLOGOUS END JOINING AND INTERACTS WITH THE KU COMPLEX

    DEFF Research Database (Denmark)

    Shamanna, Raghavendra A; Singh, Dharmendra Kumar; Lu, Huiming

    2014-01-01

    RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund......-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends...... on the activity of DNA-PK, extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ...

  11. DNA Repair by Homologous Recombination, But Not by Nonhomologous End Joining, Is Elevated in Breast Cancer Cells12

    Science.gov (United States)

    Mao, Zhiyong; Jiang, Ying; Liu, Xiang; Seluanov, Andrei; Gorbunova, Vera

    2009-01-01

    Aberrant double-stranded break (DSB) repair leads to genomic instability, which is a hallmark of malignant cells. Double-stranded breaks are repaired by two pathways: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). It is not known whether these repair pathways are affected in sporadic breast tumors. Here, we examined the efficiency of HR and NHEJ repair in a panel of sporadic breast cancer cell lines and tested whether the efficiency of HR or NHEJ correlates with radioresistance. Homologous recombination and NHEJ in breast cancer cells were analyzed using in vivo fluorescent assays. Unexpectedly, our analysis revealed that the efficiency of HR is significantly elevated in breast cancer cells compared with normal mammary epithelial cells. In contrast, the efficiency of NHEJ in breast cancer cells is not different from normal cells. Overall, breast cancer cells were more sensitive to radiation than normal cells, but the levels of resistance did not correlate with either HR or NHEJ efficiency. Thus, we demonstrate that sporadic breast cancers are not associated with a deficiency in DSB repair, but rather with upregulation of the HR pathway. Our finding of elevated HR in sporadic breast cancer cell lines suggests that therapies directed against the components of HR will be highly tumor-specific. PMID:19568413

  12. DNA Repair by Homologous Recombination, But Not by Nonhomologous End Joining, Is Elevated in Breast Cancer Cells

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    Zhiyong Mao

    2009-07-01

    Full Text Available Aberrant double-stranded break (DSB repair leads to genomic instability, which is a hallmark of malignant cells. Double-stranded breaks are repaired by two pathways: homologous recombination (HR and nonhomologous DNA end joining (NHEJ. It is not known whether these repair pathways are affected in sporadic breast tumors. Here, we examined the efficiency of HR and NHEJ repair in a panel of sporadic breast cancer cell lines and tested whether the efficiency of HR or NHEJ correlates with radioresistance. Homologous recombination and NHEJ in breast cancer cells were analyzed using in vivo fluorescent assays. Unexpectedly, our analysis revealed that the efficiency of HR is significantly elevated in breast cancer cells compared with normal mammary epithelial cells. In contrast, the efficiency of NHEJ in breast cancer cells is not different from normal cells. Overall, breast cancer cells were more sensitive to radiation than normal cells, but the levels of resistance did not correlate with either HR or NHEJ efficiency. Thus, we demonstrate that sporadic breast cancers are not associated with a deficiency in DSB repair, but rather with upregulation of the HR pathway. Our finding of elevated HR in sporadic breast cancer cell lines suggests that therapies directed against the components of HR will be highly tumor-specific.

  13. Proofreading activity of DNA polymerase Pol2 mediates 3'-end processing during nonhomologous end joining in yeast.

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    Shun-Fu Tseng

    2008-04-01

    Full Text Available Genotoxic agents that cause double-strand breaks (DSBs often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ, but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends, Pol2 is important for the recession of 3' flaps that can form during imprecise pairing. Indeed, a mutation in the 3'-5' exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3' flaps. Thus, Pol2 performs a key 3' end-processing step in NHEJ.

  14. Involvement of artemis in nonhomologous end-joining during immunoglobulin class switch recombination

    NARCIS (Netherlands)

    L. Du (Likun); M.D. van Burg; S.W. Popov (Sergey); A. Kotnis (Ashwin); J.J.M. van Dongen (Jacques); A.R. Gennery (Andrew); Q. Pan-Hammarström (Qiang)

    2008-01-01

    textabstractDNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonho- mologous end-joining (NHEJ

  15. Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells.

    Science.gov (United States)

    Bindra, Ranjit S; Goglia, Alexander G; Jasin, Maria; Powell, Simon N

    2013-06-01

    Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.

  16. Knock-in reporter mice demonstrate that DNA repair by non-homologous end joining declines with age.

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    Amita Vaidya

    2014-07-01

    Full Text Available Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs, we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age.

  17. Knock-In Reporter Mice Demonstrate that DNA Repair by Non-homologous End Joining Declines with Age

    Science.gov (United States)

    Vaidya, Amita; Mao, Zhiyong; Tian, Xiao; Spencer, Brianna; Seluanov, Andrei; Gorbunova, Vera

    2014-01-01

    Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs), we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ) pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ) events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age. PMID:25033455

  18. A DNA-PKcs mutation in a radiosensitive T-B- SCID patient inhibits Artemis activation and nonhomologous end-joining

    NARCIS (Netherlands)

    M. van der Burg (Mirjam); H. IJspeert (Hanna); N.S. Verkaik (Nicole); T. Turul (Tuba); W.W. Wiegant (Wouter); K. Morotomi-Yano (Keiko); P.O. Mari (Pierre-Olivier); I. Tezcan (Ilhan); D.J. Chen (David); M.Z. Zdzienicka (Malgorzata); J.J.M. van Dongen (Jacques); D.C. van Gent (Dik)

    2009-01-01

    textabstractRadiosensitive T-B- severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-

  19. Change of gene structure and function by non-homologous end-joining, homologous recombination, and transposition of DNA.

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    Wolfgang Goettel

    2009-06-01

    Full Text Available An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to

  20. De novo CNV formation in mouse embryonic stem cells occurs in the absence of Xrcc4-dependent nonhomologous end joining.

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    Martin F Arlt

    2012-09-01

    Full Text Available Spontaneous copy number variant (CNV mutations are an important factor in genomic structural variation, genomic disorders, and cancer. A major class of CNVs, termed nonrecurrent CNVs, is thought to arise by nonhomologous DNA repair mechanisms due to the presence of short microhomologies, blunt ends, or short insertions at junctions of normal and de novo pathogenic CNVs, features recapitulated in experimental systems in which CNVs are induced by exogenous replication stress. To test whether the canonical nonhomologous end joining (NHEJ pathway of double-strand break (DSB repair is involved in the formation of this class of CNVs, chromosome integrity was monitored in NHEJ-deficient Xrcc4(-/- mouse embryonic stem (ES cells following treatment with low doses of aphidicolin, a DNA replicative polymerase inhibitor. Mouse ES cells exhibited replication stress-induced CNV formation in the same manner as human fibroblasts, including the existence of syntenic hotspot regions, such as in the Auts2 and Wwox loci. The frequency and location of spontaneous and aphidicolin-induced CNV formation were not altered by loss of Xrcc4, as would be expected if canonical NHEJ were the predominant pathway of CNV formation. Moreover, de novo CNV junctions displayed a typical pattern of microhomology and blunt end use that did not change in the absence of Xrcc4. A number of complex CNVs were detected in both wild-type and Xrcc4(-/- cells, including an example of a catastrophic, chromothripsis event. These results establish that nonrecurrent CNVs can be, and frequently are, formed by mechanisms other than Xrcc4-dependent NHEJ.

  1. Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining.

    Science.gov (United States)

    Zong, Dali; Callén, Elsa; Pegoraro, Gianluca; Lukas, Claudia; Lukas, Jiri; Nussenzweig, André

    2015-05-26

    DNA double strand breaks (DSBs) formed during S phase are preferentially repaired by homologous recombination (HR), whereas G1 DSBs, such as those occurring during immunoglobulin class switch recombination (CSR), are repaired by non-homologous end joining (NHEJ). The DNA damage response proteins 53BP1 and BRCA1 regulate the balance between NHEJ and HR. 53BP1 promotes CSR in part by mediating synapsis of distal DNA ends, and in addition, inhibits 5' end resection. BRCA1 antagonizes 53BP1 dependent DNA end-blocking activity during S phase, which would otherwise promote mutagenic NHEJ and genome instability. Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair. Here, we ask whether increased expression of RNF168 or 53BP1 impacts physiological versus mutagenic NHEJ. We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR. As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors.

  2. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    Science.gov (United States)

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  3. Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

    Science.gov (United States)

    Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

    2007-01-01

    We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

  4. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    Energy Technology Data Exchange (ETDEWEB)

    Henrique Barreta, Marcos [Universidade Federal de Santa Catarina, Campus Universitario de Curitibanos, Curitibanos, SC (Brazil); Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Ferreira, Rogerio [Centro de Educacao Superior do Oeste-Universidade do Estado de Santa Catarina, Chapeco, SC (Brazil); Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Bordignon, Vilceu, E-mail: vilceu.bordignon@mcgill.ca [Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC (Canada)

    2012-10-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  5. COM-1 promotes homologous recombination during Caenorhabditis elegans meiosis by antagonizing Ku-mediated non-homologous end joining.

    Science.gov (United States)

    Lemmens, Bennie B L G; Johnson, Nicholas M; Tijsterman, Marcel

    2013-01-01

    Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1-deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.

  6. Inhibition of proteasomal degradation of rpn4 impairs nonhomologous end-joining repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Donghong Ju

    Full Text Available BACKGROUND: The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 induces the proteasome genes and is rapidly degraded by the assembled proteasome. The integrity of the Rpn4-proteasome feedback loop is critical for cell viability under stressed conditions. We have demonstrated that inhibition of Rpn4 degradation sensitizes cells to DNA damage, particularly in response to high doses of DNA damaging agents. The underlying mechanism, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast genetics and biochemical approach we show that inhibition of Rpn4 degradation displays a synthetic growth defect with deletion of the MEC1 checkpoint gene and sensitizes several checkpoint mutants to DNA damage. In addition, inhibition of Rpn4 degradation leads to a defect in repair of double-strand breaks (DSBs by nonhomologous end-joining (NHEJ. The expression levels of several key NHEJ genes are downregulated and the recruitment of Yku70 to a DSB is reduced by inhibition of Rpn4 degradation. We find that Rpn4 and the proteasome are recruited to a DSB, suggesting their direct participation in NHEJ. Inhibition of Rpn4 degradation may result in a concomitant delay of release of Rpn4 and the proteasome from a DSB. CONCLUSION/SIGNIFICANCE: This study provides the first evidence for the role of proteasomal degradation of Rpn4 in NHEJ.

  7. TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna.

    Science.gov (United States)

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2016-11-07

    Transcription activator-like effector nucleases (TALENs) are versatile tools that enable the insertion of DNA into different organisms. Here, we confirmed TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna, a model organism for ecological and toxicological genomics. We tested two different TALENs, ey1 TALEN and ey2 TALEN, both of which target the eyeless locus. The donor DNA plasmid, harbouring the H2B-GFP reporter gene, was designed to contain both TALEN target sites and was co-injected with each TALEN mRNA into eggs. The ey1 TALEN and ey2 TALEN constructs both resulted in H2B-GFP expression in Daphnia with a germline transmission efficiency of 3%. Of the three transgenic animals generated, two had donor DNA at the targeted genomic site, which suggested concurrent cleavage of the injected plasmid DNA and genome DNA. The availability of such tools that are capable of targeted knock-in of foreign genes will be extremely useful for advancing the knowledge of gene function and contribute to an increased understanding of functional genomics in Daphnia.

  8. The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.

    Science.gov (United States)

    Park, Jihye; Welner, Robert S; Chan, Mei-Yee; Troppito, Logan; Staber, Philipp B; Tenen, Daniel G; Yan, Catherine T

    2016-01-01

    Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients.

  9. Genetic probing of homologous recombination and non-homologous end joining during meiotic prophase in irradiated mouse spermatocytes

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    Ahmed, Emad A. [Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Department of Zoology, Faculty of Science, Assiut University, 71516 Assiut (Egypt); Philippens, Marielle E.P.; Kal, Henk B. [Department of Radiotherapy, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht (Netherlands); Rooij, Dirk G. de, E-mail: d.g.derooij@uu.nl [Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam (Netherlands); Boer, Peter de [Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen (Netherlands)

    2010-06-01

    This study was designed to obtain a better insight into the relative contribution of homologous recombination (HR) and non-homologous end joining (NHEJ) to the repair of radiation-induced DNA double-strand breaks (DSBs) at first meiotic prophase. Early and late pachytene and early diplotene spermatocytes that had completed crossing over were sampled. We studied the kinetics of {gamma}-H2AX chromatin foci removal after irradiation of mice deficient for HR and mice deficient for NHEJ. Analyzing {gamma}-H2AX signals in unirradiated RAD54/RAD54B deficient spermatocytes indicated incomplete meiotic recombination repair due to the pronounced increase of {gamma}-H2AX foci in late prophase primary spermatocytes. In these mice, 8 h after irradiation, early pachytene spermatocytes showed a reduction of the numbers of {gamma}-H2AX foci by 52% compared to 82% in the wild type, the difference being significant. However, after crossing over (in late pachytene and early diplotene), no effect of RAD54/RAD54B deficiency on the reduction of irradiation-induced foci was observed. In NHEJ deficient SCID mice, repair kinetics in early spermatocytes were similar to those in wild type mice. However, 1 h after irradiation in late pachytene and early diplotene spermatocytes 1.7 times more foci were found than in wild type mice. This difference might be related to the absence of a DNA-PKcs dependent fast repair component in SCID mice. As subsequent repair is normal, HR likely is taking over. Taken together, the results obtained in RAD54/RAD54B deficient mice and in SCID mice indicate that DSB repair in early pachytene spermatocytes is mainly carried out through HR. In late spermatocytes (late pachytenes and early diplotenes) NHEJ is active. However, probably there is an interplay between these repair pathways and when in late spermatocytes the NHEJ pathway is compromised HR may take over.

  10. SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products.

    Science.gov (United States)

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

    2014-12-01

    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-PK(cs) 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-PK(cs) 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.

  11. Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

    Science.gov (United States)

    Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

    2014-01-01

    The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date.

  12. SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products.

    Directory of Open Access Journals (Sweden)

    Gregory A Sowd

    2014-12-01

    Full Text Available 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-PK(cs 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-PK(cs 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.

  13. DNA ligase C1 mediates the LigD-independent nonhomologous end-joining pathway of Mycobacterium smegmatis.

    Science.gov (United States)

    Bhattarai, Hitesh; Gupta, Richa; Glickman, Michael S

    2014-10-01

    Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3' phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo.

  14. CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Hexi Shen

    2017-01-01

    Full Text Available Double-strand breaks (DSBs are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR and nonhomologous end-joining (NHEJ. NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ and the more error-prone KU-independent backup-NHEJ (b-NHEJ pathways, involving the poly (ADP-ribose polymerases (PARPs. However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3 and protoporphyrinogen oxidase (PPO genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80, b-NHEJ (parp1 parp2, or both (ku80 parp1 parp2. We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.

  15. Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anna Brzostek

    Full Text Available The intracellular pathogen Mycobacterium tuberculosis (Mtb is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs. These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR and non-homologous ends joining (NHEJ, in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA, NHEJ [Δ(ku,ligD], or both DSB repair systems [Δ(ku,ligD,recA]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

  16. Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Qi Jia

    2012-01-01

    Full Text Available In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

  17. Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Ding, Nan [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Qi, Yongmei; Zhang, Yingmei [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Wang, Jufang [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Huang, Dejun, E-mail: huangdj@lzu.edu.cn [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China)

    2015-09-15

    Highlights: • Cadmium (Cd) exposure delayed the repair of DNA damage induced by X-ray. • Cd exposure altered the phosphorylation of DNA-PKcs on Thr-2609 and Ser-2056 sites. • Cd impaired the formation of XRCC4 and Ligase IV foci, and down-regulated their protein expression. • Zinc mitigated the effects of Cd on DDR by regulating pDNA-PKcs (Thr-2609), XRCC4 and Ligase IV. - Abstract: Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1–8 h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.

  18. Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

    Energy Technology Data Exchange (ETDEWEB)

    Fan Jinshui; Robert, Carine [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States); Jang, Yoon-Young; Liu Hua; Sharkis, Saul; Baylin, Stephen Bruce [Johns Hopkins University School of Medicine, Department of Oncology, Baltimore, MD 21231-1000 (United States); Rassool, Feyruz Virgilia, E-mail: frassool@som.umaryland.edu [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States)

    2011-08-01

    Highlights: {yields} iPSC and hESC demonstrate a similar cell cycle profile, with increased S phase cells and decreased G0/G1. {yields} iPSC and hESC increased ROS and decreased DSBs, compared with differentiated parental cells. {yields} iPSC and hESC demonstrate elevated DSB repair activity, including nonhomologous end-joining, compared with differentiated parental cells. {yields} iPSC however show a partial apoptotic response to DNA damage, compared to hESC. {yields} DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming. - Abstract: To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to 'dedifferentiate' into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived

  19. Multiple Ku orthologues mediate DNA non-homologous end-joining in the free-living form and during chronic infection of Sinorhizobium meliloti.

    Science.gov (United States)

    Kobayashi, Hajime; Simmons, Lyle A; Yuan, Daniel S; Broughton, William J; Walker, Graham C

    2008-01-01

    The bacterial non-homologous end-joining (NHEJ) apparatus is a two-component system that uses Ku and LigD to repair DNA double-strand breaks. Although the reaction mechanism has been extensively studied, much less is known about the physiological role of bacterial NHEJ. Recent studies suggest that NHEJ acts under conditions where DNA replication is reduced or absent (such as in a spore or stationary phase). Interestingly, genes encoding Ku and LigD have been identified in a wide range of bacteria that can chronically infect eukaryotic hosts. Strikingly, Sinohizobium meliloti, an intracellular symbiont of legume plants, carries four genes encoding Ku homologues (sku1 to sku4). Deletion analysis of the sku genes indicated that all Ku homologues are functional. One of these genes, sku2, is strongly expressed in free-living cells, as well as in bacteroid cells residing inside of the host plant. To visualize the NHEJ apparatus in vivo, SKu2 protein was fused to yellow fluorescent protein (YFP). Ionizing radiation (IR) induced focus formation of SKu2-YFP in free-living cells in a dosage-dependent manner. Moreover, SKu2-YFP foci formed in response to IR in non-dividing bacteroids, indicating that NHEJ system is functional even during the chronic infection phase of symbiosis.

  20. Non-homologous end joining dependency of {gamma}-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Heidenreich, Erich [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)]. E-mail: erich.heidenreich@meduniwien.ac.at; Eisler, Herfried [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)

    2004-11-22

    There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by {gamma}-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the {gamma}-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.

  1. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

    Science.gov (United States)

    Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

    2013-12-01

    The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

  2. Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

    Science.gov (United States)

    Beckta, Jason M; Dever, Seth M; Gnawali, Nisha; Khalil, Ashraf; Sule, Amrita; Golding, Sarah E; Rosenberg, Elizabeth; Narayanan, Aarthi; Kehn-Hall, Kylene; Xu, Bo; Povirk, Lawrence F; Valerie, Kristoffer

    2015-09-29

    Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

  3. Differential role of nonhomologous end joining factors in the generation, DNA damage response, and myeloid differentiation of human induced pluripotent stem cells.

    Science.gov (United States)

    Felgentreff, Kerstin; Du, Likun; Weinacht, Katja G; Dobbs, Kerry; Bartish, Margarita; Giliani, Silvia; Schlaeger, Thorsten; DeVine, Alexander; Schambach, Axel; Woodbine, Lisa J; Davies, Graham; Baxi, Sachin N; van der Burg, Mirjam; Bleesing, Jack; Gennery, Andrew; Manis, John; Pan-Hammarström, Qiang; Notarangelo, Luigi D

    2014-06-17

    Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation, microcephaly, and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4), Artemis, and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders, we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity, but not Artemis deficiency, were associated with markedly reduced reprogramming efficiency, which could be partially rescued by genetic complementation. Moreover, we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest, particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-, but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming, with a major role for LIG4 and DNA-PKcs and a minor, if any, for Artemis.

  4. Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding.

    Science.gov (United States)

    Chiruvella, Kishore K; Renard, Brian M; Birkeland, Shanda R; Sunder, Sham; Liang, Zhuobin; Wilson, Thomas E

    2014-12-01

    LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure–function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4–Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association.

  5. Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway.

    Science.gov (United States)

    Della-Maria, Julie; Zhou, Yi; Tsai, Miaw-Sheue; Kuhnlein, Jeff; Carney, James P; Paull, Tanya T; Tomkinson, Alan E

    2011-09-30

    Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.

  6. Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions

    Science.gov (United States)

    Moeller, Ralf; Schuerger, Andrew C.; Reitz, Günther; Nicholson, Wayne L.

    2011-09-01

    Spores of Bacillus subtilis were used as a model system to study the impact of the two major DNA double-strand break (DSB) repair mechanisms [homologous recombination (HR) and non-homologous end-joining (NHEJ)] on the survivability of air-dried mono- and multilayers of bacterial spores under a simulated martian environment; i.e., an environment with low temperature (-10 °C), pure CO 2 atmosphere (99.99% CO 2), 200-1100 nm UV-VIS-NIR radiation, and 0.69 kPa pressure. Spores in multilayers exhibited low inactivation rates compared to monolayers, mainly due to shadowing effects of overlying spores. Simulated martian UV irradiation reduced dramatically spore viability, whereas when shielded from martian UV radiation, spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to simulated martian environmental conditions than were wild-type spores. In addition, NHEJ-deficient spores were consistently more sensitive than HR-deficient spores to simulated Mars environmental conditions, suggesting that DSBs were an important type of DNA damage. The results indicated that both HR and NHEJ provide an efficient set of DNA repair pathways ensuring spore survival after exposure to simulated martian environmental conditions.

  7. Efficient gene targeting in ligase IV-deficient Monascus ruber M7 by perturbing the non-homologous end joining pathway.

    Science.gov (United States)

    He, Yi; Shao, Yanchun; Chen, Fusheng

    2014-01-01

    Inactivating the non-homologous end joining (NHEJ) pathway is a well established method to increase gene replacement frequency (GRF) in filamentous fungi because NHEJ is predominant for the repair of DNA double strand breaks (DSBs), while gene targeting is based on homologous recombination (HR). DNA ligase IV, a component of the NHEJ system, is strictly required for the NHEJ in Saccharomyces cerevisiae and Neurospora crassa. To enhance the GRF in Monascus ruber M7, we deleted the Mrlig4 gene encoding a homolog of N. crassa DNA ligase IV. The obtained mutant (MrΔlig4) showed no apparent defects in vegetative growth, colony phenotype, microscopic morphology, spore yield, and production of Monascus pigments and citrinin compared with the wild-type strain (M. ruber M7). Gene targeting of ku70 and triA genes revealed that GRF in the MrΔlig4 strain increased four-fold compared with that in the wild-type strain, reached 68 % and 85 %, respectively. Thus, the MrΔlig4 strain is a promising host for efficient genetic manipulation. In addition, the MrΔlig4 strain is more sensitive than M. ruber M7 to a DNA-damaging agent, methyl methanesulfonate.

  8. Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells.

    Science.gov (United States)

    Renouf, Benjamin; Soret, Christine; Saandi, Thoueiba; Delalande, François; Martin, Elisabeth; Vanier, Marie; Duluc, Isabelle; Gross, Isabelle; Freund, Jean-Noël; Domon-Dell, Claire

    2012-04-01

    Cdx2, a gene of the paraHox cluster, encodes a homeodomain transcription factor that plays numerous roles in embryonic development and in homeostasis of the adult intestine. Whereas Cdx2 exerts a tumor suppressor function in the gut, its abnormal ectopic expression in acute leukemia is associated to a pro-oncogenic function. To try to understand this duality, we have hypothesized that Cdx2 may interact with different protein partners in the two tissues and set up experiments to identify them by tandem affinity purification. We show here that Cdx2 interacts with the Ku heterodimer specifically in intestinal cells, but not in leukemia cells, via its homeodomain. Ku proteins do not affect Cdx2 transcriptional activity. However, Cdx2 inhibits in vivo and in vitro the DNA repair activity mediated by Ku proteins in intestinal cells. Whereas Cdx2 does not affect the recruitment of Ku proteins and DNA-PKcs into the DNA repair complex, it inhibits DNA-PKcs activity. Thus, we report here a new function of Cdx2, acting as an inhibitor of the DNA repair machinery, that may contribute to its tumor suppressor function specifically in the gut.

  9. Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Farjana Fattah

    2010-02-01

    Full Text Available The repair of DNA double-strand breaks (DSBs is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR and non-homologous end joining (NHEJ. In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways-the main Ku heterodimer-dependent or "classic" NHEJ (C-NHEJ pathway and an "alternative" NHEJ (A-NHEJ pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PK(cs, XLF, and LIGIV, and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PK(cs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PK(cs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

  10. An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.

    Science.gov (United States)

    Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K; Chokshi, Chirayu; Tsai, Miaw-Sheue; Matsumoto, Yoshihiro; Kuzdovich, Monica; Remesh, Soumya G; Fang, Shujuan; Tomkinson, Alan E; Lees-Miller, Susan P; Tainer, John A

    2016-12-30

    DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

  11. Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS

    Directory of Open Access Journals (Sweden)

    De Benedetti Arrigo

    2011-01-01

    Full Text Available Abstract Background The S. cerevisiae mating type switch model of double-strand break (DSB repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ. We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin. Findings We previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1 The site had become less accessible to cleavage by the HO endonuclease; 2 the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3 occupancy of nucleosomes at the site (ChIP for histone H3 was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created

  12. Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Campos-Nebel, Marcelo de [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)], E-mail: mnebel@hematologia.anm.edu.ar; Larripa, Irene; Gonzalez-Cid, Marcela [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)

    2008-11-10

    Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by {gamma}H2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU

  13. Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

    Directory of Open Access Journals (Sweden)

    Stenerlöw Bo

    2007-10-01

    Full Text Available Abstract Background Efficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood. Results In this report we demonstrate that long single-stranded DNA (ssDNA ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times ≥ 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G1-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE, no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50°C are used. Conclusion We provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required.

  14. Investigations on the role of base excision repair and non-homologous end-joining pathways in sodium selenite-induced toxicity and mutagenicity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Mániková, Dominika; Vlasáková, Danusa; Loduhová, Jana; Letavayová, Lucia; Vigasová, Dana; Krascsenitsová, Eva; Vlcková, Viera; Brozmanová, Jela; Chovanec, Miroslav

    2010-03-01

    Selenium (Se) belongs to nutrients that are essential for human health. Biological activity of this compound, however, mainly depends on its dose, with a potential of Se to induce detrimental effects at high doses. Although mechanisms lying behind detrimental effects of Se are poorly understood yet, they involve DNA damage induction. Consequently, DNA damage response and repair pathways may play a crucial role in cellular response to Se. Using Saccharomyces cerevisiae we showed that sodium selenite (SeL), an inorganic form of Se, can be toxic and mutagenic in this organism due to its ability to induce DNA double-strand breaks (DSBs). Moreover, we reported that a spectrum of mutations induced by this compound in the stationary phase of growth is mainly represented by 1-4 bp deletions. Consequently, we proposed that SeL acts as an oxidizing agent in yeast producing oxidative damage to DNA. As short deletions could be anticipated to arise as a result of action of non-homologous end-joining (NHEJ) and oxidative damage to DNA is primarily coped with base excision repair (BER), a contribution of these two pathways towards survival, DSB induction, mutation frequency and types of mutations following SeL exposure was examined in present study. First, we show that while NHEJ plays no role in repairing toxic DNA lesions induced by SeL, cells with impairment in BER are sensitized towards this compound. Of BER activities examined, those responsible for processing of 3'-blocking DNA termini seem to be the most crucial for manifestation of the toxic effects of SeL in yeast. Second, an impact of NHEJ and BER on DSB induction after SeL exposure turned to be inappreciable, as no increase in DNA double-strand breakage in NHEJ and BER single or NHEJ BER double mutant upon SeL exposure was observed. Finally, we demonstrate that impairment in both these pathways does not importantly change mutation frequency after SeL exposure and that NHEJ is not responsible for generation of short

  15. Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate nonhomologous DNA end joining: relevance to cancer, aging,and the immune system

    Institute of Scientific and Technical Information of China (English)

    Michael R Lieber; Haihui Lu; Jiafeng Gu; Klaus Schwarz

    2008-01-01

    Nonhomologous DNA end joining (NHEJ) is the primary pathway for repair of double-strand DNA breaks in hu-man cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect ad-ditional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configura-tions reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual com-ponents also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.

  16. A Role for BLM in Double-Strand Break Repair Pathway Choice: Prevention of CtIP/Mre11-Mediated Alternative Nonhomologous End-Joining

    DEFF Research Database (Denmark)

    Grabarz, Anastazja; Guirouilh-Barbat, Josée; Barascu, Aurelia;

    2013-01-01

    The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp) generated by alternative end-joining (A-EJ). BLM...... represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role...... for BLM that influences the DSB repair pathway choice: (1) protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2) promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid...

  17. A Role for BLM in Double-Strand Break Repair Pathway Choice: Prevention of CtIP/Mre11-Mediated Alternative Nonhomologous End-Joining

    Directory of Open Access Journals (Sweden)

    Anastazja Grabarz

    2013-10-01

    Full Text Available The choice of the appropriate double-strand break (DSB repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp generated by alternative end-joining (A-EJ. BLM represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role for BLM that influences the DSB repair pathway choice: (1 protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2 promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid unscheduled resection that might jeopardize genome integrity.

  18. Alternative end-joining mechanisms: a historical perspective

    Directory of Open Access Journals (Sweden)

    Anabelle eDecottignies

    2013-04-01

    Full Text Available In the presence of functional DNA repair pathways, DNA double-strand breaks (DSBs are mainly repaired by non-homologous end-joining (NHEJ or homologous recombination (HR, two conserved pathways that protect cells from aberrant chromosomal rearrangements. During the past two decades however, unusual and presumably distinct DNA end-joining repair activities have been unraveled in NHEJ-deficient cells and these are likely to operate in various chromosomal contexts and species. Most alternative DNA end-joining events reported so far appear to involve microhomologous sequences and are likely to rely on a subset of HR enzymes, namely those responsible for the single-strand annealing mechanism of HR, and on DNA Ligase III. Usually, microhomologies are not initially present at DSB ends and thus need to be unmasked through DNA end resection, a process that can lead to extensive nucleotide loss and is therefore highly mutagenic. In addition to microhomology-mediated end-joining events, recent studies in mammalian cells point towards the existence of a distinct and still ill defined alternative end-joining pathway that does not appear to rely on pre-existing microhomologies and may possibly involve DNA Ligase I.Whether dependent on microhomologies or not, alternative DNA end-joining mechanisms are likely to be highly mutagenic in vivo, being able to drive telomere fusion events and cancer-associated chromosomal translocations in mouse models. In the future, it will be important to better characterize the genetic requirements of these mutagenic alternative mechanisms of DNA end-joining.

  19. Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila.

    Directory of Open Access Journals (Sweden)

    Sze Ham Chan

    2010-07-01

    Full Text Available DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or "alternative" end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta, encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.

  20. Human transcriptional coactivator PC4 stimulates DNA end joining and activates DSB repair activity.

    Science.gov (United States)

    Batta, Kiran; Yokokawa, Masatoshi; Takeyasu, Kunio; Kundu, Tapas K

    2009-01-23

    Human transcriptional coactivator PC4 is a highly abundant nuclear protein that is involved in diverse cellular processes ranging from transcription to chromatin organization. Earlier, we have shown that PC4, a positive activator of p53, overexpresses upon genotoxic insult in a p53-dependent manner. In the present study, we show that PC4 stimulates ligase-mediated DNA end joining irrespective of the source of DNA ligase. Pull-down assays reveal that PC4 helps in the association of DNA ends through its C-terminal domain. In vitro nonhomologous end-joining assays with cell-free extracts show that PC4 enhances the joining of noncomplementary DNA ends. Interestingly, we found that PC4 activates double-strand break (DSB) repair activity through stimulation of DSB rejoining in vivo. Together, these findings demonstrate PC4 as an activator of nonhomologous end joining and DSB repair activity.

  1. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  2. CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.

    Science.gov (United States)

    Lee-Theilen, Mieun; Matthews, Allysia J; Kelly, Dierdre; Zheng, Simin; Chaudhuri, Jayanta

    2011-01-01

    Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized physiological role of microhomology-mediated end joining in a C-NHEJ-proficient environment.

  3. Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity.

    Science.gov (United States)

    Kirkman, Laura A; Lawrence, Elizabeth A; Deitsch, Kirk W

    2014-01-01

    Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite's genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites.

  4. Alternative end-joining pathway(s): bricolage at DNA breaks.

    Science.gov (United States)

    Frit, Philippe; Barboule, Nadia; Yuan, Ying; Gomez, Dennis; Calsou, Patrick

    2014-05-01

    To cope with DNA double strand break (DSB) genotoxicity, cells have evolved two main repair pathways: homologous recombination which uses homologous DNA sequences as repair templates, and non-homologous Ku-dependent end-joining involving direct sealing of DSB ends by DNA ligase IV (Lig4). During the last two decades a third player most commonly named alternative end-joining (A-EJ) has emerged, which is defined as any Ku- or Lig4-independent end-joining process. A-EJ increasingly appears as a highly error-prone bricolage on DSBs and despite expanding exploration, it still escapes full characterization. In the present review, we discuss the mechanism and regulation of A-EJ as well as its biological relevance under physiological and pathological situations, with a particular emphasis on chromosomal instability and cancer. Whether or not it is a genuine DSB repair pathway, A-EJ is emerging as an important cellular process and understanding A-EJ will certainly be a major challenge for the coming years.

  5. The Relationship between Radiosensitivity and DNA Double Strand Breaks Repair Genes in Non-homologous End Joining Pathway in Human Cervical Carcinoma%宫颈癌组织LIG4、XRCC4、XRCC5、XRCC6、XRCC7的mRNA表达及与放疗敏感性的关系

    Institute of Scientific and Technical Information of China (English)

    张宇卉; 折虹; 王艳阳; 何剑莉; 海平; 刘长虎

    2013-01-01

    目的 研究DNA双链断裂非同源末端链接(NHEJ)修复通路中DNA连接酶IV(LIG4)、X射线修复交叉互补基因4、5、6、7(XRCC4、XRCC5、XRCC6、XRCC7)的mRNA表达水平与宫颈癌组织放射敏感性的关系.方法 应用实时荧光定量RT-PCR技术检测61例宫颈癌组织中的LIG4、XRCC4、XRCC5、XRCC6和XRCC7的mRNA表达量,依据WHO实体瘤疗效评定标准,将放疗后的宫颈癌患者分为放疗高敏感组和放疗低敏感组,对比两组间以上基因表达水平的差异.结果 61例宫颈癌患者放疗后疗效评价为完全缓解(CR):60.7%(37/61),部分缓解及无变化(PR+NC):39.3%(24/61).据此分为宫颈癌放疗高敏感组(CR)与放疗低敏感组(PR+NC).放疗高敏感组LIG4mRNA、XRCC4mRNA、XRCC5mRNA、XRCC6mRNA、XRCC7mRNA在宫颈癌组织中的表达分别为0.84±0.60、1.46±0.58、2.07±1.09、0.54±0.23和0.60±0.34;放疗低敏感组为1.30±0.50、2.08±0.75、2.96±1.07、0.72±0.17和0.90±0.27,两组比较差异均有统计学意义(t=2.54、3.24、2.70、2.73、2.97,P=0.014、0.002、0.009、0.008、0.004).结论 患者宫颈癌组织中LIG4、XRCC4、XRCC5、XRCC6和XRCC7的mRNA高表达者,其对放疗抗拒,低表达则放疗高度敏感,上述基因可能成为预测宫颈癌放疗敏感性的指标和放疗增敏的分子靶点.%Objective To investigate the correlation between expression levels of the five key genes in Non -homologous End Joining Pathway( NHEJ ) and radiosensitivity in cervical carcinoma tissues. Methods The mRNA expression levels of LIG4, XRCC4, XRCC5 , XRCC6, XRCC7 were detected by real - time quantitative reverse transcription - polymerase chain reaction ( QRT - PCR ) method in 61 cervical carcinoma tissues. According to the World Health Orgnization solid tumor response judgment standard, the patients with cervical carcinoma were divided into two groups,which were high radiosensitive and low radiosensitive. The mRNA expressions of LIG4,XRCC4,XRCC5 ,XRCC6,XRCC7 were

  6. Unsolved mystery: the role of BRCA1 in DNA end-joining.

    Science.gov (United States)

    Saha, Janapriya; Davis, Anthony J

    2016-08-01

    Heritable mutations in the tumor suppressor gene BRCA1 increase a woman's lifetime risk of developing breast and ovarian cancer. BRCA1's tumor suppressor function is directly linked to its myriad of functions in the cellular response to DNA double-strand breaks (DSBs). BRCA1 interacts with an extensive array of DNA damage responsive proteins and plays important roles in DSB repair, mediated by the homologous recombination pathway, and in the activation of cell cycle checkpoints. However, the role of BRCA1 in the other two DSB repair pathways, classical non-homologous end-joining (C-NHEJ) and alternative NHEJ (A-NHEJ), remains unclear. In this review, we will discuss the current literature on BRCA1's potential role(s) in modulating both C-NHEJ and A-NHEJ. We also present a model showing that BRCA1 contributes to genomic maintenance by promoting precise DNA repair across all cell cycle phases via the direct modulation of DNA end-joining.

  7. DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?

    OpenAIRE

    Weterings, Eric; Chen, David J.

    2007-01-01

    The DNA-dependent protein kinase (DNA-PK) is one of the central enzymes involved in DNA double-strand break (DSB) repair. It facilitates proper alignment of the two ends of the broken DNA molecule and coordinates access of other factors to the repair complex. We discuss the latest findings on DNA-PK phosphorylation and offer a working model for the regulation of DNA-PK during DSB repair.

  8. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

    Directory of Open Access Journals (Sweden)

    Aurélie Kapusta

    2011-04-01

    Full Text Available During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs, each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs, which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ, are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new

  9. Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.

    Directory of Open Access Journals (Sweden)

    Kishore K Chiruvella

    2013-06-01

    Full Text Available DNA ligase IV (Dnl4 in budding yeast is a specialized ligase used in non-homologous end joining (NHEJ of DNA double-strand breaks (DSBs. Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5' resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.

  10. Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.

    Science.gov (United States)

    Chiruvella, Kishore K; Liang, Zhuobin; Birkeland, Shanda R; Basrur, Venkatesha; Wilson, Thomas E

    2013-06-01

    DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5' resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.

  11. Human RECQ1 interacts with Ku70/80 and modulates DNA end-joining of double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Swetha Parvathaneni

    Full Text Available Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome or premature aging (Werner Syndrome. RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.

  12. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining.

    Science.gov (United States)

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L; Tomkinson, Alan E; Tainer, John A; Ellenberger, Tom

    2015-08-18

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.

  13. Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination.

    Science.gov (United States)

    Masani, Shahnaz; Han, Li; Meek, Katheryn; Yu, Kefei

    2016-02-02

    Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

  14. DNA ligase III and DNA ligase IV carry out genetically distinct forms of end joining in human somatic cells.

    Science.gov (United States)

    Oh, Sehyun; Harvey, Adam; Zimbric, Jacob; Wang, Yongbao; Nguyen, Thanh; Jackson, Pauline J; Hendrickson, Eric A

    2014-09-01

    Ku-dependent C-NHEJ (classic non-homologous end joining) is the primary DNA EJing (end joining) repair pathway in mammals. Recently, an additional EJing repair pathway (A-NHEJ; alternative-NHEJ) has been described. Currently, the mechanism of A-NHEJ is obscure although a dependency on LIGIII (DNA ligase III) is often implicated. To test the requirement for LIGIII in A-NHEJ we constructed a LIGIII conditionally-null human cell line using gene targeting. Nuclear EJing activity appeared unaffected by a deficiency in LIGIII as, surprisingly, so were random gene targeting integration events. In contrast, LIGIII was required for mitochondrial function and this defined the gene's essential activity. Human Ku:LIGIII and Ku:LIGIV (DNA ligase IV) double knockout cell lines, however, demonstrated that LIGIII is required for the enhanced A-NHEJ activity that is observed in Ku-deficient cells. Most unexpectedly, however, the majority of EJing events remained LIGIV-dependent. In conclusion, although human LIGIII has an essential function in mitochondrial maintenance, it is dispensable for most types of nuclear DSB repair, except for the A-NHEJ events that are normally suppressed by Ku. Moreover, we describe that a robust Ku-independent, LIGIV-dependent repair pathway exists in human somatic cells.

  15. Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining

    DEFF Research Database (Denmark)

    Zong, Dali; Callén, Elsa; Pegoraro, Gianluca;

    2015-01-01

    BP1 and BRCA1 regulate the balance between NHEJ and HR. 53BP1 promotes CSR in part by mediating synapsis of distal DNA ends, and in addition, inhibits 5' end resection. BRCA1 antagonizes 53BP1 dependent DNA end-blocking activity during S phase, which would otherwise promote mutagenic NHEJ and genome...

  16. 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination.

    Science.gov (United States)

    Bothmer, Anne; Robbiani, Davide F; Feldhahn, Niklas; Gazumyan, Anna; Nussenzweig, Andre; Nussenzweig, Michel C

    2010-04-12

    Class switch recombination (CSR) diversifies antibodies by joining highly repetitive DNA elements, which are separated by 60-200 kbp. CSR is initiated by activation-induced cytidine deaminase, an enzyme that produces multiple DNA double-strand breaks (DSBs) in switch regions. Switch regions are joined by a mechanism that requires an intact DNA damage response and classical or alternative nonhomologous end joining (A-NHEJ). Among the DNA damage response factors, 53BP1 has the most profound effect on CSR. We explore the role of 53BP1 in intrachromosomal DNA repair using I-SceI to introduce paired DSBs in the IgH locus. We find that the absence of 53BP1 results in an ataxia telangiectasia mutated-dependent increase in DNA end resection and that resected DNA is preferentially repaired by microhomology-mediated A-NHEJ. We propose that 53BP1 favors long-range CSR in part by protecting DNA ends against resection, which prevents A-NHEJ-dependent short-range rejoining of intra-switch region DSBs.

  17. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    Science.gov (United States)

    Roth, D B; Wilson, J H

    1985-05-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-base-pair duplication at its termini. Once inside the cell, this molecule must circularize to initiate lytic infection. Circularization can occur either by direct, nonhomologous end-joining or by homologous recombination within the duplicated region. Although the products of the two recombination pathways are different, they are equally infectious. Since homologous and nonhomologous recombination processes are competing for the same substrate, the relative amounts of the products of each pathway should reflect the relative rates of homologous and nonhomologous recombination. Analysis of individual recombinant genomes from 164 plaques indicates that the rate of circularization by nonhomologous recombination is 2- to 3-fold higher than the rate of homologous recombination. The assay system described here may prove to be useful for testing procedures designed to influence the relative rates of homologous and nonhomologous recombination.

  18. Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions.

    Science.gov (United States)

    Konomura, Naoto; Arai, Naoto; Shinohara, Takeshi; Kobayashi, Jun; Iwasaki, Wakana; Ikawa, Shukuko; Kusano, Kohji; Shibata, Takehiko

    2017-01-09

    RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.

  19. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining

    Science.gov (United States)

    van Schendel, Robin; van Heteren, Jane; Welten, Richard; Tijsterman, Marcel

    2016-01-01

    For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs). This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3’ overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles. PMID:27755535

  20. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining.

    Directory of Open Access Journals (Sweden)

    Robin van Schendel

    2016-10-01

    Full Text Available For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS and photo-activated trimethylpsoralen (UV/TMP, that deletion mutagenesis is the result of polymerase Theta (POLQ-mediated end joining (TMEJ of double strand breaks (DSBs. This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3' overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles.

  1. SIRT1 inhibition impairs non-homologous end joining DNA damage repair by increasing Ku70 acetylation in chronic myeloid leukemia cells.

    Science.gov (United States)

    Zhang, Wenjun; Wu, Haixia; Yang, Meng; Ye, Shiguang; Li, Liang; Zhang, Hong; Hu, Jiong; Wang, Xuguang; Xu, Jun; Liang, Aibin

    2016-03-22

    Most chemotherapeutic agents for leukemia are DNA damaging agents. However, DNA lesions can be repaired by activities of DNA repair systems. Increasing evidence have shown that enhanced DNA damage repair capacity contributes to chemotherapy resistance in leukemia cells. Thus, targeting DNA repair mechanisms is a promising strategy for novel leukemia treatment. SIRT1 expressions were downregulated by lentivirus-delivered SIRT1 shRNA in myeloid leukemia cells. SIRT1 mRNA and protein levels were analyzed by real-time PCR and Western blot, respectively. Flow cytometry was carried out to analyze cell cycle progression, apoptosis and DNA damage repair efficiency. DNA damage levels were assessed by alkaline comet assay, and H2AX phosphorylation was analyzed by immunoblotting and immunofluorescence. A mouse leukemia model was established by transplanting lentivirus-infected K562 cells containing SIRT1 shRNA into sublethally irradiated NOD/SCID mice, and tumorigenesis was evaluated by detecting tumor weights and mice survival. SIRT1 expressions were upregulated in myeloid leukemic patients. Downregulation of SIRT1 by RNAi promoted etoposide-induced DNA damage in myeloid leukemia cells accompanied by reduced NHEJ activity, and increased Ku70 acetylation. Furthermore, SIRT1 knockdown resulted in cell cycle arrest, induction of apoptosis and reduction of K562 cell proliferation accompanied by enhanced p53 and FOXO1 acetylation in K562 cells after etoposide treatment. Importantly, SIRT1 downregulation reduced the tumorigenesis ability of K562 cells in mouse xenografts following chemotherapy treatment. These results revealed that SIRT1 promotes the NHEJ repair pathway by deacetylating Ku70 in K562 cells, suggesting that SIRT1 is a novel therapeutic target for treating myeloid leukemia.

  2. Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways.

    Science.gov (United States)

    Sears, Catherine R; Turchi, John J

    2012-07-13

    The treatment for advanced stage non-small cell lung cancer (NSCLC) often includes platinum-based chemotherapy and IR. Cisplatin and IR combination therapy display schedule and dose-dependent synergy, the mechanism of which is not completely understood. In a series of in vitro and cell culture assays in a NSCLC model, we investigated both the downstream and direct treatment and damage effects of cisplatin on NHEJ catalyzed repair of a DNA DSB. The results demonstrate that extracts prepared from cisplatin-treated cells are fully capable of NHEJ catalyzed repair of a DSB using a non-cisplatin-damaged DNA substrate in vitro. Similarly, using two different host cell reactivation assays, treatment of cells prior to transfection of a linear, undamaged reporter plasmid revealed no reduction in NHEJ compared with untreated cells. In contrast, transfection of a linear GFP-reporter plasmid containing site-specific, cisplatin lesions 6-bp from the termini revealed a significant impairment in DSB repair of the cisplatin-damaged DNA substrates in the absence of cellular treatment with cisplatin. Together, these data demonstrate that impaired NHEJ in combined cisplatin-IR treated cells is likely the result of a direct effect of cisplatin-DNA lesions near a DSB and that the indirect cellular effects of cisplatin treatment are not significant contributors to the synergistic cytotoxicity observed with combination cisplatin-IR treatment.

  3. Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end joining

    NARCIS (Netherlands)

    Kobayashi, S.; Kasaishi, Y.; Nakada, S.; Takagi, T.; Era, S.; Motegi, A.; Chiu, R. K.; Takeda, S.; Hirota, K.

    2015-01-01

    The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at.-ray-induced DSB sites. To analyze functional interactions between the three e

  4. Impaired 53BP1/RIF1 DSB mediated end-protection stimulates CtIP-dependent end resection and switches the repair to PARP1-dependent end joining in G1.

    Science.gov (United States)

    Bakr, Ali; Köcher, Sabrina; Volquardsen, Jennifer; Petersen, Cordula; Borgmann, Kerstin; Dikomey, Ekkehard; Rothkamm, Kai; Mansour, Wael Y

    2016-09-06

    End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.

  5. Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism.

    Science.gov (United States)

    Laffitte, Marie-Claude N; Leprohon, Philippe; Hainse, Maripier; Légaré, Danielle; Masson, Jean-Yves; Ouellette, Marc

    2016-06-01

    The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability.

  6. Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism.

    Directory of Open Access Journals (Sweden)

    Marie-Claude N Laffitte

    2016-06-01

    Full Text Available The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability.

  7. Constitutional Chromothripsis Rearrangements Involve Clustered Double-Stranded DNA Breaks and Nonhomologous Repair Mechanisms

    Directory of Open Access Journals (Sweden)

    Wigard P. Kloosterman

    2012-06-01

    Full Text Available Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements.

  8. Histone deacetylase inhibitors selectively target homology dependent DNA repair defective cells and elevate non-homologous endjoining activity.

    Directory of Open Access Journals (Sweden)

    Stephanie Smith

    Full Text Available BACKGROUND: We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi trichostatin A (TSA, confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity. RESULTS: HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR factor BLM or the non-homologous end-joining (NHEJ and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency. CONCLUSIONS: HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks

  9. Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

    Science.gov (United States)

    Lloyd, Andrew H; Wang, Dong; Timmis, Jeremy N

    2012-01-01

    DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

  10. DNA双链断裂NHEJ修复及其与肿瘤的研究%Non-homologous end joining pathway of DSB repair and cancer

    Institute of Scientific and Technical Information of China (English)

    张耀伟

    2010-01-01

    非同源末端连接是哺乳动物最主要的DNA双链断裂(DSB)连接方式.肿瘤细胞非同源末端连接能力的提高与其放化疗抵抗有关,抑制肿瘤细胞非同源末端连接能力,可能增加其对放化疗的敏感性.因此,参与非同源末端连接的修复因子可能成为肿瘤分子靶向治疗及放化疗增敏的新治疗点.%Non-homologous end joining (NHEJ) is the major pathway for repairing DNA doublestrand break (DSB) in mammalian species. The capacity of NHEJ increases in tumor cell,which plays a role in radiation/chemotherapy-resistant agent Inhibiting DSB rejoining may play a crucial role in the enhancement of cellular radiation/chemotherapy-sensitizing. Thus, the protein molecule enrolled in NHEJ may be new potential targets for radiation/chemotherapy -sensitizing.

  11. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.

    Science.gov (United States)

    Nakade, Shota; Tsubota, Takuya; Sakane, Yuto; Kume, Satoshi; Sakamoto, Naoaki; Obara, Masanobu; Daimon, Takaaki; Sezutsu, Hideki; Yamamoto, Takashi; Sakuma, Tetsushi; Suzuki, Ken-ichi T

    2014-11-20

    Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

  12. Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context.

    Science.gov (United States)

    Gunn, Amanda; Bennardo, Nicole; Cheng, Anita; Stark, Jeremy M

    2011-12-09

    During repair of multiple chromosomal double strand breaks (DSBs), matching the correct DSB ends is essential to limit rearrangements. To investigate the maintenance of correct end use, we examined repair of two tandem noncohesive DSBs generated by endonuclease I-SceI and the 3' nonprocessive exonuclease Trex2, which can be expressed as an I-SceI-Trex2 fusion. We examined end joining (EJ) repair that maintains correct ends (proximal-EJ) versus using incorrect ends (distal-EJ), which provides a relative measure of incorrect end use (distal end use). Previous studies showed that ATM is important to limit distal end use. Here we show that DNA-PKcs kinase activity and RAD50 are also important to limit distal end use, but that H2AX is dispensable. In contrast, we find that ATM, DNA-PKcs, and RAD50 have distinct effects on repair events requiring end processing. Furthermore, we developed reporters to examine the effects of the transcription context on DSB repair, using an inducible promoter. We find that a DSB downstream from an active promoter shows a higher frequency of distal end use, and a greater reliance on ATM for limiting incorrect end use. Conversely, DSB transcription context does not affect end processing during EJ, the frequency of homology-directed repair, or the role of RAD50 and DNA-PKcs in limiting distal end use. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing.

  13. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

    Science.gov (United States)

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M; Samuel, Glady Hazitha; Anderson, Michelle A E; Dahlem, Timothy J; Myles, Kevin M; Adelman, Zach N

    2015-03-31

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

  14. Increasing the efficiency of homologous recombination vec-tor-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts%siRNA 干扰绵羊胚胎成纤维细胞 Lig4基因增加同源重组载体重连修复效率

    Institute of Scientific and Technical Information of China (English)

    王伟; 王玉霜; 黄兰兰; 简子健; 王新华; 刘守仁; 皮文辉

    2016-01-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to ef-fectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene pro-vides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts.%在动物细胞中,抑制非同源末端连接(Non-homologous end joining, NHEJ)修复途径,可以提高同源重组(Homologous recombination, HR)修复基因组双链断裂(Double-strand brakes, DSBs)的发生概率。为了提高绵羊胚胎成纤维细胞的 HR 效率,针对 NHEJ 修复途径中的关键因子 Lig4(DNA ligase 4)基因,本文设计合成4个具有靶向性的 siRNA(Small interfering RNA)。绵羊胚胎成纤维细胞经电转染导入 siRNA,通过实时荧光定量PCR(qRT-PCR)和 Western blotting 检测,筛选出有效抑制 Lig4基因表达的2个 siRNA。应用质粒重连法检测HR 修复效率,将 I-SceⅠ酶线性化的 HR 质粒和 siRNA 共转染绵羊胚胎成纤维细胞,经72 h 培养及流式细胞仪检测,与对照

  15. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    OpenAIRE

    Roth, D B; Wilson, J H

    1985-01-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-ba...

  16. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair.

    Science.gov (United States)

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-01

    Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

  17. Deletion of exon 8 from the EXT1 gene causes multiple osteochondromas (MO) in a family with three affected members.

    Science.gov (United States)

    Zhuang, Lei; Gerber, Simon D; Kuchen, Stefan; Villiger, Peter M; Trueb, Beat

    2016-01-01

    Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

  18. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  19. Non-homologous isofunctional enzymes: A systematic analysis of alternative solutions in enzyme evolution

    Directory of Open Access Journals (Sweden)

    Wolf Yuri I

    2010-04-01

    Full Text Available Abstract Background Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (nonhomologous relationships between proteins. Results We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. Conclusions These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity. Reviewers This article was reviewed by Andrei

  20. Homologous and non-homologous recombination differentially affect DNA damage repair in mice.

    NARCIS (Netherlands)

    J. Essers (Jeroen); H. van Steeg (Harry); J. de Wit (Jan); M. Vermeij (Marcel); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland); S.M.A. Swagemakers (Sigrid)

    2000-01-01

    textabstractIonizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the a

  1. Human nucleolus organizers on nonhomologous chromosomes can share the same ribosomal gene variants.

    OpenAIRE

    Krystal, M; D'Eustachio, P; Ruddle, F H; Arnheim, N

    1981-01-01

    The distributions of three human ribosomal gene polymorphisms among individual chromosomes containing nucleolus organizers were analyzed by using mouse--human hybrid cells. Different nucleolus organizers can contain the same variant, suggesting the occurrence of genetic exchanges among ribosomal gene clusters on nonhomologous chromosomes. Such exchanges appear to occur less frequently in mice. This difference is discussed in terms of the nucleolar organization and chromosomal location of ribo...

  2. Extensive nonhomologous meiotic synapsis between normal chromosome axes of an rcp(3;6)(p14;q21) translocation in a hairless Mexican boar.

    Science.gov (United States)

    Villagómez, D A F; Ayala-Valdovinos, M A; Galindo-García, J; Sánchez-Chipres, D R; Mora-Galindo, J; Taylor-Preciado, J J

    2008-01-01

    Due to its low fertility, expressed as small litter size, a Mexican hairless boar was subjected to cytogenetic investigation. Analysis of G-banded mitotic chromosomes revealed a reciprocal chromosome translocation, rcp(3;6) (p14;q21). Synaptonemal complex analysis showed a regular pairing behavior of the translocation chromosome axes, always resulting in a quadrivalent configuration. However, due to extensive nonhomologous pairing between the axes of nonderivative chromosomes 3 and 6, the quadrivalent mostly had an asymmetrical cross-shaped morphology. The nonhomologous pairing occurred not only at mid and late pachytene, but also at the earliest stage of pachytene. It seems that early pachytene heterosynapsis is a common phenomenon in the pairing behavior of pig reciprocal translocations. Therefore, heterosynapsis may reduce apoptosis of germ cells due to partial absence of homologous synapsis during the pairing phase of meiosis. The frequency of spermatocytes showing quadrivalent configurations with unpaired axial segments apparently did not affect germ cell progression in the boar, since fairly normal testicular histology was noticed.

  3. Productive homologous and non-homologous recombination of hepatitis C virus in cell culture

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Galli, Andrea; Li, Yi-Ping

    2013-01-01

    . In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a......) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13-36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6...

  4. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    Science.gov (United States)

    Lin, Yu-Fen; Nagasawa, Hatsumi; Little, John B; Kato, Takamitsu A; Shih, Hung-Ying; Xie, Xian-Jin; Wilson, Paul F; Brogan, John R; Kurimasa, Akihiro; Chen, David J; Bedford, Joel S; Chen, Benjamin P C

    2014-01-01

    We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  5. Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Didelot Xavier

    2012-06-01

    Full Text Available Abstract Background Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved. Results We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E. From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E, which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination. Conclusions Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and

  6. Bloom syndrome radials are predominantly non-homologous and are suppressed by phosphorylated BLM.

    Science.gov (United States)

    Owen, Nichole; Hejna, James; Rennie, Scott; Mitchell, Asia; Hanlon Newell, Amy; Ziaie, Navid; Moses, Robb E; Olson, Susan B

    2014-01-01

    Biallelic mutations in BLM cause Bloom syndrome (BS), a genome instability disorder characterized by growth retardation, sun sensitivity and a predisposition to cancer. As evidence of decreased genome stability, BS cells demonstrate not only elevated levels of spontaneous sister chromatid exchanges (SCEs), but also exhibit chromosomal radial formation. The molecular nature and mechanism of radial formation is not known, but radials have been thought to be DNA recombination intermediates between homologs that failed to resolve. However, we find that radials in BS cells occur over 95% between non-homologous chromosomes, and occur non-randomly throughout the genome. BLM must be phosphorylated at T99 and T122 for certain cell cycle checkpoints, but it is not known whether these modifications are necessary to suppress radial formation. We find that exogenous BLM constructs preventing phosphorylation at T99 and T122 are not able to suppress radial formation in BS cells, but are able to inhibit SCE formation. These findings indicate that BLM functions in 2 distinct pathways requiring different modifications. In one pathway, for which the phosphorylation marks appear dispensable, BLM functions to suppress SCE formation. In a second pathway, T99 and T122 phosphorylations are essential for suppression of chromosomal radial formation, both those formed spontaneously and those formed following interstrand crosslink damage.

  7. Msh2 blocks an alternative mechanism for non-homologous tail removal during single-strand annealing in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Glenn M Manthey

    Full Text Available Chromosomal translocations are frequently observed in cells exposed to agents that cause DNA double-strand breaks (DSBs, such as ionizing radiation and chemotherapeutic drugs, and are often associated with tumors in mammals. Recently, translocation formation in the budding yeast, Saccharomyces cerevisiae, has been found to occur at high frequencies following the creation of multiple DSBs adjacent to repetitive sequences on non-homologous chromosomes. The genetic control of translocation formation and the chromosome complements of the clones that contain translocations suggest that translocation formation occurs by single-strand annealing (SSA. Among the factors important for translocation formation by SSA is the central mismatch repair (MMR and homologous recombination (HR factor, Msh2. Here we describe the effects of several msh2 missense mutations on translocation formation that suggest that Msh2 has separable functions in stabilizing annealed single strands, and removing non-homologous sequences from their ends. Additionally, interactions between the msh2 alleles and a null allele of RAD1, which encodes a subunit of a nuclease critical for the removal of non-homologous tails suggest that Msh2 blocks an alternative mechanism for removing these sequences. These results suggest that Msh2 plays multiple roles in the formation of chromosomal translocations following acute levels of DNA damage.

  8. A mediator methylation mystery: JMJD1C demethylates MDC1 to regulate DNA repair.

    Science.gov (United States)

    Lu, Jian; Matunis, Michael J

    2013-12-01

    Mediator of DNA-damage checkpoint 1 (MDMDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation and sumoylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDMDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

  9. Genomic rearrangements at the FRA2H common fragile site frequently involve non-homologous recombination events across LTR and L1(LINE) repeats.

    Science.gov (United States)

    Brueckner, Lena M; Sagulenko, Evgeny; Hess, Elisa M; Zheglo, Diana; Blumrich, Anne; Schwab, Manfred; Savelyeva, Larissa

    2012-08-01

    Common fragile sites (cFSs) are non-random chromosomal regions that are prone to breakage under conditions of replication stress. DNA damage and chromosomal alterations at cFSs appear to be critical events in the development of various human diseases, especially carcinogenesis. Despite the growing interest in understanding the nature of cFS instability, only a few cFSs have been molecularly characterised. In this study, we fine-mapped the location of FRA2H using six-colour fluorescence in situ hybridisation and showed that it is one of the most active cFSs in the human genome. FRA2H encompasses approximately 530 kb of a gene-poor region containing a novel large intergenic non-coding RNA gene (AC097500.2). Using custom-designed array comparative genomic hybridisation, we detected gross and submicroscopic chromosomal rearrangements involving FRA2H in a panel of 54 neuroblastoma, colon and breast cancer cell lines. The genomic alterations frequently involved different classes of long terminal repeats and long interspersed nuclear elements. An analysis of breakpoint junction sequence motifs predominantly revealed signatures of microhomology-mediated non-homologous recombination events. Our data provide insight into the molecular structure of cFSs and sequence motifs affected by their activation in cancer. Identifying cFS sequences will accelerate the search for DNA biomarkers and targets for individualised therapies.

  10. The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells.

    Science.gov (United States)

    Amornwichet, Napapat; Oike, Takahiro; Shibata, Atsushi; Nirodi, Chaitanya S; Ogiwara, Hideaki; Makino, Haruhiko; Kimura, Yuka; Hirota, Yuka; Isono, Mayu; Yoshida, Yukari; Ohno, Tatsuya; Kohno, Takashi; Nakano, Takashi

    2015-06-11

    Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.

  11. Removal of nonhomologous DNA ends in double-strand break recombination: The role of the yeast ultraviolet repair gene RAD1

    Energy Technology Data Exchange (ETDEWEB)

    Fishman-Lobell, J.; Habert, J.E. (Brandeis Univ., Waltham, MA (United States))

    1992-10-15

    Double-strand breaks (DSBs) in Saccharomyces cerevisiae can be repaired by gene conversions or by deletions resulting from single-strand annealing between direct repeats of homologous sequences. Although rad1 mutants are resistant to x-rays and can complete DSB-mediated mating-type switching, they could not complete recombination when the ends of the break contained approximately 60 base pairs of nonhomology. Recombination was restored when the ends of the break were made homologous to donor sequences. Additionally, the absence of RAD1 led to the frequent appearance of a previously unobserved type of recombination product. These data suggest RAD1 is required to remove nonhomologous DNA from the 3{prime} ends of recombining DNA, a process analogous to the excision of photodimers during repair of ultraviolet-damaged DNA.

  12. A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.

    Science.gov (United States)

    Dong, Biao; Moore, Andrea R; Dai, Jihong; Roberts, Sean; Chu, Kirk; Kapranov, Philipp; Moss, Bernard; Xiao, Weidong

    2013-07-01

    Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.

  13. Ultraviolet sensitivity of the addition, deletion and replacement of long nonhomologous DNA segments by genetic transformation of Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Walter, R.B.; Stuy, J.H. (Florida State Univ., Tallahassee (USA). Dept. of Biological Science)

    1982-03-01

    The construction and some properties of Haemophilus influenzae Rd strains with long and different R plasmid-derived DNA segments (nonhomologous inserts) at the same site in the HP1 prophage have previously been described. These inserts can be added to a recipient's genome by genetic transformation, they can be deleted from the recipient genome, or they can be replaced by another insert. It is reported that the UV inactivation of all three phenomena followed single hit kinetics. Deletion was roughly 10 times more resistant; its UV-sensitivity equalled that of a high-efficiency point mutation. There was an inverse correlation between UV-sensitivity and additive transformation efficiency of the various inserts; sensitivity may thus be a measure of insert size. This correlation was not seen for deletion. All three phenomena were more sensitive when they were measured on excision repair-deficient uvr/sup -/ recipients. The dose-reduction factor for addition was about 1.5 while it was about 2.6 for deletion.

  14. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

    Science.gov (United States)

    Nishizawa-Yokoi, Ayako; Cermak, Tomas; Hoshino, Tomoki; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Starker, Colby; Voytas, Daniel F; Toki, Seiichi

    2016-02-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.

  15. Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

    OpenAIRE

    2013-01-01

    Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-hom...

  16. Telomeric trans-silencing in Drosophila melanogaster: tissue specificity, development and functional interactions between non-homologous telomeres.

    Directory of Open Access Journals (Sweden)

    Thibaut Josse

    Full Text Available BACKGROUND: The study of P element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE, a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, "TAS" has the capacity to repress in trans, in the female germline, a homologous P-lacZ transgene located in euchromatin. TSE can show variegation in ovaries, displays a maternal effect as well as an epigenetic transmission through meiosis and involves heterochromatin and RNA silencing pathways. PRINCIPAL FINDINGS: Here, we analyze phenotypic and genetic properties of TSE. We report that TSE does not occur in the soma at the adult stage, but appears restricted to the female germline. It is detectable during development at the third instar larvae where it presents the same tissue specificity and maternal effect as in adults. Transgenes located in TAS at the telomeres of the main chromosomes can be silencers which in each case show the maternal effect. Silencers located at non-homologous telomeres functionally interact since they stimulate each other via the maternally-transmitted component. All germinally-expressed euchromatic transgenes tested, located on all major chromosomes, were found to be repressed by a telomeric silencer: thus we detected no TSE escaper. The presence of the euchromatic target transgene is not necessary to establish the maternal inheritance of TSE, responsible for its epigenetic behavior. A single telomeric silencer locus can simultaneously repress two P-lacZ targets located on different chromosomal arms. CONCLUSIONS AND SIGNIFICANCE: Therefore TSE appears to be a widespread phenomenon which can involve different telomeres and work across the genome. It can explain the P cytotype establishment by telomeric P elements in natural Drosophila populations.

  17. Evolution of bacterial phosphoglycerate mutases: non-homologous isofunctional enzymes undergoing gene losses, gains and lateral transfers.

    Directory of Open Access Journals (Sweden)

    Jeremy M Foster

    Full Text Available BACKGROUND: The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms. METHODS/PRINCIPAL FINDINGS: To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect. CONCLUSIONS/SIGNIFICANCE: Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non

  18. Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji, E-mail: kzsuzuki@nagasaki-u.ac.jp [Course of Life Sciences and Radiation Research, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Kodama, Seiji [Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-machi, Sakai 599-8570 (Japan); Watanabe, Masami [Kyoto University Research Reactor Institute, Kumatori-cho Sennan-gun, Osaka 590-0494 (Japan)

    2010-01-05

    Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined. We found that delayed induction of DNA double strand breaks and chromosomal breaks were intact in Ku80-deficient cells surviving X-irradiation, whereas there was no sign for the production of chromosome bridges between divided daughter cells. Moreover, delayed induction of dicentric chromosomes was significantly compromised in those cells compared to the wild-type CHO cells. Reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and delayed cell death, indicating that defective Ku80-dependent dicentric induction was the cause of the absence of delayed cell death. Since DNA-PKcs-defective cells showed delayed phenotypes, Ku80-dependent illegitimate rejoining is involved in delayed impairment of the integrity of the genome in radiation-survived cells.

  19. CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle

    OpenAIRE

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

    The repair of DNA double-strand breaks (DSB) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSB occurs through non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSB by homologous recomb...

  20. Evaluation of DNA Repair Function as a Predictor of Response in a Clinical Trial of PARP Inhibitor Monotherapy for Recurrent Ovarian Carcinoma

    Science.gov (United States)

    2014-10-01

    DNA ligase IV, XRCC4 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC...nonhomologous end-joining (NHEJ) pathway (53BP1, Ku70, Ku80, DNA-PKcs, XRCC4, DNA ligase IV) as well as PARP1. This group of proteins was chosen based on our...recombination, nonhomologous end-joining (NHEJ), immunohistochemistry, poly(ADP-ribose) polymerase, Ku70, Ku80, PARP1, 53BP1, DNA-PK, Artemis, DNA ligase IV

  1. Affective Urbanism

    DEFF Research Database (Denmark)

    Samson, Kristine

    Urban design and architecture are increasingly used as material and affective strategies for setting the scene, for manipulation and the production of urban life: The orchestration of atmospheres, the framing and staging of urban actions, the programming for contemplation, involvement, play, expe...... affects can be choreographed and designed intentionally or whether it arises from unpredictable circumstances within urbanity itself....

  2. Affective Maps

    DEFF Research Database (Denmark)

    Salovaara-Moring, Inka

    of environmental knowledge production. It uses InfoAmazonia, the databased platform on Amazon rainforests, as an example of affective geo-visualization within information mapping that enhances embodiment in the experience of the information. Amazonia is defined as a digitally created affective (map)space within...

  3. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); J.H.J. Hoeijmakers (Jan); D.C. van Gent (Dik)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  4. Training Program in the Molecular Basis of Breast Cancer Research

    Science.gov (United States)

    2000-08-01

    activities of Rad5l viable, no growth defects binding of DSB ends Rad52-1- ES cells show a moderate reduction in homologous recombination frequency Rad54... recombination frequency and are sensitive to IR, sister chromatid exchange MMS, and mitomycin C V(D)J recombination and non-homologous end-joining

  5. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  6. A robust network of double-strand break repair pathways governs genome integrity during C. elegans development.

    NARCIS (Netherlands)

    Pontier, D.B.; Tijsterman, M.

    2009-01-01

    To preserve genomic integrity, various mechanisms have evolved to repair DNA double-strand breaks (DSBs). Depending on cell type or cell cycle phase, DSBs can be repaired error-free, by homologous recombination, or with concomitant loss of sequence information, via nonhomologous end-joining (NHEJ) o

  7. Analysis of DNA double-strand break repair pathways in mice

    Energy Technology Data Exchange (ETDEWEB)

    Brugmans, Linda [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Kanaar, Roland [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands); Essers, Jeroen [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands) and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands)]. E-mail: j.essers@erasmusmc.nl

    2007-01-03

    During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.

  8. Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status.

    NARCIS (Netherlands)

    Sprong, D.; Janssen, H.L.K.; Vens, C.; Begg, A.C.

    2006-01-01

    PURPOSE: To determine the role of DNA repair in hypoxic radioresistance. METHODS AND MATERIALS: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (&

  9. A new type of radiosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation

    NARCIS (Netherlands)

    M. van der Burg (Mirjam); M.Z. Zdzienicka (Malgorzata); D.C. van Gent (Dik); J.J.M. van Dongen (Jacques); L.R. van Veelen (Lieneke); N.S. Verkaik (Nicole); W.W. Wiegant (Wouter); N.G. Hartwig (Nico); B.H. Barendregt (Barbara); L.J.L. Brugmans (Linda); A. Raams (Anja); N.G.J. Jaspers (Nicolaas)

    2006-01-01

    textabstractV(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of

  10. DNA Polymerase θ: Duct Tape and Zip Ties for a Fragile Genome.

    Science.gov (United States)

    Willis, Nicholas A; Scully, Ralph

    2016-08-18

    Using a combination of genetics and cellular DNA rejoining assays, in this issue of Molecular Cell, Wyatt et al. (2016) demonstrate a critical role for mammalian DNA polymerase θ in the rejoining of DNA ends that are poor substrates for classical non-homologous end joining.

  11. Differential regulation of the cellular response to DNA double-strand breaks in G1

    DEFF Research Database (Denmark)

    Barlow, Jacqueline H; Lisby, Michael; Rothstein, Rodney

    2008-01-01

    Double-strand breaks (DSBs) are potentially lethal DNA lesions that can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that DSBs induced by ionizing radiation (IR) are efficiently processed for HR and bound by Rfa1 during G1, while endonuclease-in...

  12. Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency

    NARCIS (Netherlands)

    K. Felgentreff (Kerstin); Y.N. Lee (Yu Nee); F. Frugoni (Francesco); L. Du (Likun); M. van der Burg (Mirjam); S. Giliani; I. Tezcan (Ilhan); I. Reisli (Ismail); E. Mejstříková (Ester); J.P. de Villartay; B.P. Sleckman (Barry P.); J. Manis (John); L.D. Notarangelo (Luigi Daniele)

    2015-01-01

    textabstractBackground The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficien

  13. Crypt base columnar stem cells in small intestines of mice are radioresistant

    NARCIS (Netherlands)

    Hua, G.; Thin, T.H.; Feldman, R.; Haimovitz-Friedman, A.; Clevers, H.; Fuks, Z.; Kolesnick, R.

    2012-01-01

    BACKGROUND & AIMS: Adult stem cells have been proposed to be quiescent and radiation resistant, repairing DNA double-strand breaks by nonhomologous end joining. However, the population of putative small intestinal stem cells (ISCs) at position +4 from the crypt base contradicts this model, in that t

  14. Deletion of a KU80 homolog enhances homologous recombination in the thermotolerant yeast Kluyveromyces marxianus.

    Science.gov (United States)

    Choo, Jin Ho; Han, Changpyo; Kim, Jae-Young; Kang, Hyun Ah

    2014-10-01

    Targeted gene replacement in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555 has been hampered by its propensity to non-homologous end joining (NHEJ). To enhance homologous recombination (HR) by blocking NHEJ, we identified and disrupted the K. marxianus KU80 gene. The ku80 deletion mutant strain (Kmku80∆) of K. marxianus KCTC 17555 did not show apparent growth defects under several conditions with the exception of exposure to tunicamycin. The targeted disruption of the three model genes, KmLEU2, KmPDC1, and KmPDC5, was increased by 13-70 % in Kmku80∆, although the efficiency was greatly affected by the length of the homologous flanking fragments. In contrast, the double HR frequency was 0-13.7 % in the wild-type strain even with flanking fragments 1 kb long. Therefore, Kmku80∆ promises to be a useful recipient strain for targeted gene manipulation.

  15. The RSC and INO80 chromatin-remodeling complexes in DNA double-strand break repair.

    Science.gov (United States)

    Chambers, Anna L; Downs, Jessica A

    2012-01-01

    In eukaryotes, DNA is packaged into chromatin and is therefore relatively inaccessible to DNA repair enzymes. In order to perform efficient DNA repair, ATP-dependent chromatin-remodeling enzymes are required to alter the chromatin structure near the site of damage to facilitate processing and allow access to repair enzymes. Two of the best-studied remodeling complexes involved in repair are RSC (Remodels the Structure of Chromatin) and INO80 from Saccharomyces cerevisiae, which are both conserved in higher eukaryotes. RSC is very rapidly recruited to breaks and mobilizes nucleosomes to promote phosphorylation of H2A S129 and resection. INO80 enrichment at a break occurs later and is dependent on phospho-S129 H2A. INO80 activity at the break site also facilitates resection. Consequently, both homologous recombination and nonhomologous end-joining are defective in rsc mutants, while subsets of these repair pathways are affected in ino80 mutants.

  16. Human longevity and variation in DNA damage response and repair

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike

    2014-01-01

    others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning......DNA-damage response and repair are crucial to maintain genetic stability, and are consequently considered central to aging and longevity. Here, we investigate whether this pathway overall associates to longevity, and whether specific sub-processes are more strongly associated with longevity than...... and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10-5), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using...

  17. Affect Regulation

    DEFF Research Database (Denmark)

    Pedersen, Signe Holm; Poulsen, Stig Bernt; Lunn, Susanne

    2014-01-01

    Gergely and colleagues’ state that their Social Biofeedback Theory of Parental Affect Mirroring” can be seen as a kind of operationalization of the classical psychoanalytic concepts of holding, containing and mirroring. This article examines to what extent the social biofeedback theory of parenta...

  18. Affective Networks

    Directory of Open Access Journals (Sweden)

    Jodi Dean

    2010-02-01

    Full Text Available This article sets out the idea of affective networks as a constitutive feature of communicative capitalism. It explores the circulation of intensities in contemporary information and communication networks, arguing that this circulation should be theorized in terms of the psychoanalytic notion of the drive. The article includes critical engagements with theorists such as Guy Debord, Jacques Lacan, Tiziana Terranova, and Slavoj Zizek.

  19. Mechanisms of intron gain and loss in Drosophila

    Directory of Open Access Journals (Sweden)

    Yenerall Paul

    2011-12-01

    Full Text Available Abstract Background It is widely accepted that orthologous genes have lost or gained introns throughout evolution. However, the specific mechanisms that generate these changes have proved elusive. Introns are known to affect nearly every level of gene expression. Therefore, understanding their mechanism of evolution after their initial fixation in eukaryotes is pertinent to understanding the means by which organisms develop greater regulation and complexity. Results To investigate possible mechanisms of intron gain and loss, we identified 189 intron gain and 297 intron loss events among 11 Drosophila species. We then investigated these events for signatures of previously proposed mechanisms of intron gain and loss. This work constitutes the first comprehensive study into the specific mechanisms that may generate intron gains and losses in Drosophila. We report evidence of intron gain via transposon insertion; the first intron loss that may have occurred via non-homologous end joining; intron gains via the repair of a double strand break; evidence of intron sliding; and evidence that internal or 5' introns may not frequently be deleted via the self-priming of reverse transcription during mRNA-mediated intron loss. Our data also suggest that the transcription process may promote or result in intron gain. Conclusion Our findings support the occurrence of intron gain via transposon insertion, repair of double strand breaks, as well as intron loss via non-homologous end joining. Furthermore, our data suggest that intron gain may be enabled by or due to transcription, and we shed further light on the exact mechanism of mRNA-mediated intron loss.

  20. [Affective dependency].

    Science.gov (United States)

    Scantamburlo, G; Pitchot, W; Ansseau, M

    2013-01-01

    Affective dependency is characterized by emotional distress (insecure attachment) and dependency to another person with a low self-esteem and reassurance need. The paper proposes a reflection on the definition of emotional dependency and the confusion caused by various denominations. Overprotective and authoritarian parenting, cultural and socio-environmental factors may contribute to the development of dependent personality. Psychological epigenetic factors, such as early socio-emotional trauma could on neuronal circuits in prefronto-limbic regions that are essential for emotional behaviour.We also focus on the interrelations between dependent personality, domestic violence and addictions. The objective for the clinician is to propose a restoration of self-esteem and therapeutic strategies focused on autonomy.

  1. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

    OpenAIRE

    Nakade, Shota; Tsubota, Takuya; Sakane, Yuto; Kume, Satoshi; Sakamoto, Naoaki; Obara, Masanobu; Daimon, Takaaki; Sezutsu, Hideki; Yamamoto, Takashi; Sakuma, Tetsushi; Ken-ichi T. Suzuki

    2014-01-01

    Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspace...

  2. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

    OpenAIRE

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M.; Samuel, Glady Hazitha; Anderson, Michelle A.E.; Dahlem, Timothy J.; Myles, Kevin M.; Adelman, Zach N.

    2015-01-01

    Mosquitoes are vectors of both parasites and viruses responsible for high-impact diseases including malaria, dengue, and chikungunya. Novel interventions based on genetic modification of the mosquito genome are currently being developed and implemented. To comprehensively exploit such interventions, detailed knowledge of mosquito physiology, genetics, and genome engineering are required. We developed and validated a two-step process for performing high-efficiency site-specific insertion of ge...

  3. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

    2013-01-01

    -PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation...... when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA...

  4. A New Era of Genome Integration-Simply Cut and Paste!

    Science.gov (United States)

    Liu, Zihe; Liang, Youyun; Ang, Ee Lui; Zhao, Huimin

    2017-01-24

    Genome integration is a powerful tool in both basic and applied biological research. However, traditional genome integration, which is typically mediated by homologous recombination, has been constrained by low efficiencies and limited host range. In recent years, the emergence of homing endonucleases and programmable nucleases has greatly enhanced integration efficiencies and allowed alternative integration mechanisms such as nonhomologous end joining and microhomology-mediated end joining, enabling integration in hosts deficient in homologous recombination. In this review, we will highlight recent advances and breakthroughs in genome integration methods made possible by programmable nucleases, and their new applications in synthetic biology and metabolic engineering.

  5. Nonhomologous Mechanisms of Repair of Chromosomal Breaks

    Energy Technology Data Exchange (ETDEWEB)

    Haber, J. E.

    2001-12-19

    Discovered three new proteins involved in DNA damage assessment. Interestingly they are all proteins involved in recombination, but they have very different roles in that process and other proteins that might be expected to be equivalently involved are not. This is developing into a very significant area of research.

  6. Repair Pathway Choices and Consequences at the Double-Strand Break.

    Science.gov (United States)

    Ceccaldi, Raphael; Rondinelli, Beatrice; D'Andrea, Alan D

    2016-01-01

    DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including genomic instability and cell death. Indeed, misrepair of DSBs can lead to inappropriate end-joining events, which commonly underlie oncogenic transformation due to chromosomal translocations. Typically, cells employ two main mechanisms to repair DSBs: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). In addition, alternative error-prone DSB repair pathways, namely alternative end joining (alt-EJ) and single-strand annealing (SSA), have been recently shown to operate in many different conditions and to contribute to genome rearrangements and oncogenic transformation. Here, we review the mechanisms regulating DSB repair pathway choice, together with the potential interconnections between HR and the annealing-dependent error-prone DSB repair pathways.

  7. Experiencing affective interactive art

    NARCIS (Netherlands)

    Bialoskorski, Leticia S.S.; Westerink, Joyce H.D.M.; Broek, van den Egon L.

    2010-01-01

    The progress in the field of affective computing enables the realization of affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on the integration of a framework for affective move

  8. Genome Editing in Escherichia coli with Cas9 and synthetic CRISPRs

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Ze; Richardson, Sarah; Robinson, David; Deutsch, Samuel; Cheng, Jan-Fang

    2014-03-14

    Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

  9. Real-time monitoring of RAG-catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex

    OpenAIRE

    Wang, Guannan; Dhar, Kajari; Swanson, Patrick C.; Levitus, Marcia; Chang, Yung

    2012-01-01

    During V(D)J recombination, the RAG1/2 recombinase is thought to play an active role in transferring newly excised recombination ends from the RAG post-cleavage complex (PCC) to the non-homologous end joining (NHEJ) machinery to promote appropriate antigen receptor gene assembly. However, this transfer mechanism is poorly understood, partly because of the technical difficulty in revealing weak association of coding ends (CEs) with one of the PCCs, coding end complex (CEC). Using fluorescence ...

  10. Radiosensitive Severe Combined Immunodeficiency Disease

    OpenAIRE

    Dvorak, Christopher C.; Cowan, Morton J.

    2010-01-01

    Inherited defects in components of the non-homologous end joining DNA repair mechanism produce a T-B-NK+ severe combined immunodeficiency disease (SCID) characterized by heightened sensitivity to ionizing radiation. Patients with the radiosensitive form of SCID may also have increased short- and long-term sensitivity to the alkylator-based chemotherapy regimens traditionally utilized for conditioning prior to allogeneic hematopoietic cell transplantation (HCT). Known etiologies of radiosensit...

  11. Mechanisms of Resistance to Chemotherapies Targeting BRCA-Mutant Breast Cancer

    Science.gov (United States)

    2015-12-01

    genetic manipulation of the major players that mediate homologous recombination and non-homologous end joining DNA repair pathways. These studies will...excessive spreading of either endogenous 53BP1 brought on by increased RNF168- mediated H2A ubiquitylati on or its exogenously overexpress ed...cells BRCA1- mediated removal of 53BP1 is thought to be an essential prerequisite for end resection and RAD51-dependent HR. In agreement

  12. To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

    OpenAIRE

    2014-01-01

    Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, ...

  13. Sir protein–independent repair of dicentric chromosomes in Saccharomyces cerevisiae

    Science.gov (United States)

    McCleary, David F.; Steakley, David Lee; Rine, Jasper

    2016-01-01

    Sir2 protein has been reported to be recruited to dicentric chromosomes under tension, and such chromosomes are reported to be especially vulnerable to breakage in sir2Δ mutants. We found that the loss of viability in such mutants was an indirect effect of the repression of nonhomologous end joining in Sir− mutants and that the apparent recruitment of Sir2 protein to chromosomes under tension was likely due to methodological weakness in early chromatin immunoprecipitation studies. PMID:27466318

  14. Sir protein-independent repair of dicentric chromosomes in Saccharomyces cerevisiae.

    Science.gov (United States)

    McCleary, David F; Steakley, David Lee; Rine, Jasper

    2016-09-15

    Sir2 protein has been reported to be recruited to dicentric chromosomes under tension, and such chromosomes are reported to be especially vulnerable to breakage in sir2Δ mutants. We found that the loss of viability in such mutants was an indirect effect of the repression of nonhomologous end joining in Sir(-) mutants and that the apparent recruitment of Sir2 protein to chromosomes under tension was likely due to methodological weakness in early chromatin immunoprecipitation studies.

  15. Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9

    Science.gov (United States)

    Iwata, Satoru; Yoshina, Sawako; Suehiro, Yuji; Hori, Sayaka; Mitani, Shohei

    2016-01-01

    Balancer chromosomes are convenient tools used to maintain lethal mutations in heterozygotes. We established a method for engineering new balancers in C. elegans by using the CRISPR/Cas9 system in a non-homologous end-joining mutant. Our studies will make it easier for researchers to maintain lethal mutations and should provide a path for the development of a system that generates rearrangements at specific sites of interest to model and analyse the mechanisms of action of genes. PMID:27650892

  16. Histone H1 functions as a stimulatory factor in backup pathways of NHEJ

    Science.gov (United States)

    Rosidi, Bustanur; Wang, Minli; Wu, Wenqi; Sharma, Aparna; Wang, Huichen; Iliakis, George

    2008-01-01

    DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ. PMID:18250087

  17. Affectivity and race

    DEFF Research Database (Denmark)

    of the Nordic countries, Affectivity and Race draws on a variety of sources, including television programmes, news media, fictional literature, interviews, ethnographic observations, teaching curricula and policy documents, to explore the ways in which ideas about affectivity and emotion afford new insights...... into the experience of racial difference and the unfolding of political discourses on race in various social spheres. Organised around the themes of the politicisation of race through affect, the way that race produces affect and the affective experience of race, this interdisciplinary collection sheds light...... on the role of feelings in the formation of subjectivities, how race and whiteness are affectively circulated in public life and the ways in which emotions contribute to regimes of inclusion and exclusion. As such it will appeal to scholars across the social sciences, with interests in sociology, anthropology...

  18. TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

    Science.gov (United States)

    Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

    2016-02-01

    Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle.

  19. The role of DNA damage and repair in decitabine-mediated apoptosis in multiple myeloma.

    Science.gov (United States)

    Maes, Ken; De Smedt, Eva; Lemaire, Miguel; De Raeve, Hendrik; Menu, Eline; Van Valckenborgh, Els; McClue, Steve; Vanderkerken, Karin; De Bruyne, Elke

    2014-05-30

    DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) are under investigation for the treatment of cancer, including the plasma cell malignancy multiple myeloma (MM). Evidence exists that DNA damage and repair contribute to the cytotoxicity mediated by the DNMTi decitabine. Here, we investigated the DNA damage response (DDR) induced by decitabine in MM using 4 human MM cell lines and the murine 5T33MM model. In addition, we explored how the HDACi JNJ-26481585 affects this DDR. Decitabine induced DNA damage (gamma-H2AX foci formation), followed by a G0/G1- or G2/M-phase arrest and caspase-mediated apoptosis. JNJ-26481585 enhanced the anti-MM effect of decitabine both in vitro and in vivo. As JNJ-26481585 did not enhance decitabine-mediated gamma-H2AX foci formation, we investigated the DNA repair response towards decitabine and/or JNJ-26481585. Decitabine augmented RAD51 foci formation (marker for homologous recombination (HR)) and/or 53BP1 foci formation (marker for non-homologous end joining (NHEJ)). Interestingly, JNJ-26481585 negatively affected basal or decitabine-induced RAD51 foci formation. Finally, B02 (RAD51 inhibitor) enhanced decitabine-mediated apoptosis. Together, we report that decitabine-induced DNA damage stimulates HR and/or NHEJ. JNJ-26481585 negatively affects RAD51 foci formation, thereby providing an additional explanation for the combinatory effect between decitabine and JNJ-26481585.

  20. Colors Can Affect Us!

    Institute of Scientific and Technical Information of China (English)

    张俊斌

    2006-01-01

    Different colors affect us differently.The following will show us how they work. Experiment proves that math problems worked on yellow paper have fewer mistakes than problems written on other colors of paper.

  1. Pain, Affect, and Attachment

    Directory of Open Access Journals (Sweden)

    Carl Eduard Scheidt

    2015-03-01

    Full Text Available Various psychodynamic processes may underlie the development of psychogenic pain disorder such as conversion, the displacement of affect, or narcissistic defenses. However, many of the processes suggested are related to a disorder of affect regulation. The term affect regulation in psychoanalytic literature refers to phenomena which are often described by the concept of alexithymia. Empirical observations suggest that alexithymia is correlated to insecure attachment, especially an insecure dismissing representation of attachment. Psychodynamic psychotherapy in psychogenic pain disorder should focus on the reintegration of split-off affects which may provoke intensive counter-transference and which in order to be used therapeutically must be linked to attachment experiences within and outside of the therapeutic relationship.

  2. Affects and assemblages

    DEFF Research Database (Denmark)

    Samson, Kristine

    and cultural geopgraphy. On this backdrop the paper states that affects and assemblages could serve as key notions for the reassembling the aesthetics of urban space. Thus, the paper suggest a less formal understanding of urban space and aesthetics, proposing an understanding of aesthetics......Affects and assemblages are Deleuzian Guattarian notions related to aesthetics and spatial territories. In recent urban geography and urban studies these notions are increasingly gaining more impact (Amin & Thrift 2002, Pile 2008, Farías & Bender 2010, Andersen & Harrison 2010, Thrift 2008). What...... happens to aesthetics and how does it change the existing social and geographical understanding of urban space? The paper sets out to reintroduce aesthetical aspects of affects and assemblages in relation to urban space and urban planning. It presupposes urban space as a continuous state of becoming where...

  3. The Affective Turn

    Directory of Open Access Journals (Sweden)

    Alí Lara

    2013-11-01

    Full Text Available In the last decade studies on affect and emotions have become relevant in the social sciences. This is not just a fad guideline, but instead a simultaneous reader of public life changes and subjective experience, from which it is also being transformed the knowledge production. Such a trend has been known as ‘The Affective Turn’ within the Anglophone Academy. Here we will translate it as ‘El Giro Afectivo’. This turn, so far, has not dabbled in the social science literature that is written in Spanish. This paper draws on a singular panorama of discussions about contemporary social studies of affect and emotion, and it’s vertebrate by some of its expressions in the contemporary academy.

  4. Factors affecting soil cohesion

    Science.gov (United States)

    Soil erodibility is a measure of a soil’s resistance against erosive forces and is affected by both intrinsic (or inherent) soil property and the extrinsic condition at the time erodibility measurement is made. Since soil erodibility is usually calculated from results obtained from erosion experimen...

  5. How Body Affects Brain.

    Science.gov (United States)

    Suzuki, Wendy A

    2016-08-09

    Studies show that physical exercise can affect a range of brain and cognitive functions. However, little is known about the peripheral signals that initiate these central changes. Moon et al. (2016) provide exciting new evidence that a novel myokine, cathepsin B (CTSB), released with exercise is associated with improved memory.

  6. Affective World Literature

    DEFF Research Database (Denmark)

    Vilslev, Annette Thorsen

    , the dissertation investigates the critical negotiation of the novel as a travelling genre in Japan in the beginning of the 20th century, and, more specifically, Sōseki’s work in relation to world literature and affect theory. Sōseki’s work is highly influential in Japan and East Asia, and his novels widely...

  7. Affective Factors: Anxiety

    Science.gov (United States)

    Tasnimi, Mahshad

    2009-01-01

    Affective factors seem to play a crucial role in success or failure in second language acquisition. Negative attitudes can reduce learners' motivation and harm language learning, while positive attitudes can do the reverse. Discovering students' attitudes about language will help both teacher and student in teaching learning process. Anxiety is…

  8. Affective Tourism Ethnography

    NARCIS (Netherlands)

    Buda, Dorina; Tucker, Hazel

    2015-01-01

    In this paper we aim to advance the concept of affective tourism ethnography. We take ethnography to refer to a research strategy rather than simply a methodological tool. This is because ethnography entails methods (individual interviews, focus groups, participant observation amongst others) and co

  9. Individual Differences in Affect.

    Science.gov (United States)

    Haviland, Jeannette

    This paper argues that infants' affect patterns are innate and are meaningful indicators of individual differences in internal state. Videotapes of seven infants' faces were coded using an ethogram; the movement of the eyebrow, eye direction, eye openness, mouth shape, mouth position, lip position, and tongue protrusion were assessed…

  10. Dynamic Synchronization of Teacher-Students Affection in Affective Instruction

    Science.gov (United States)

    Zhang, Wenhai; Lu, Jiamei

    2011-01-01

    Based on Bower's affective network theory, the article links the dynamic analysis of affective factors in affective instruction, and presents affective instruction strategic of dynamic synchronization between teacher and students to implement the best ideal mood that promotes students' cognition and affection together. In the process of teaching,…

  11. Neurotransmitters affecting time perception

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND:It has been demonstrated that dopamine and acetylcholine are the main neurotransmitters that affect time perception,which is also affected by other neurotransmitters.OBJECTIVE:To summarize how the neurotransmitter affect the time perception,and put forward the perspectives for further study on time perception.RETRIEVE STRATEGY:An online search for related literatures published in English was conducted in Elsevier SDOL(ScienceDirect Online)database from May 1990 to March 2007 using key words of "timing neurotransmitter".Totally 69 literatures were collected,and they were primarily checked.Inclusive criteria:Reviews and experimental studies;correlative studies of timing neurotransmitter.Exclusive criteria:Repeated studies.LITERATURE EVALUATION:The literatures were mainly sourced from Cognitive Brain Research and Neuroscience,and they were analyzed according to the inclusive criteria.Nineteen of them were involved,and all were experimental studies and reviews.DATA SYNTHESIS:The studies on time perception are developed mainly concentrating on dopamine and acetylcholine.Dopamine D2 receptors mainly affect the speed of internal clock.Dopamine receptors play an important role in both timing excitation and inhibition,which suggests the bi-directional regulation of dopamine.Injection of dopamine agonist can affect the attention to timing information.Injection of BW813U(antagonist of acetylcholine) can induce memory disorder,which indicates the effect of acetylcholine on timing memory,and further study shows that it is the effect of acetylcholine in precentral medial area.In a word,the study on the neurotransmitters affecting time perception is still at the primary stage.CONCLUSION:Dopamine and acetylcholine are the neurotransmitters known to be related to time perception.Dopamine in the basal ganglia is related to internal-clock in the range of seconds and minutes;Acetylcholine in prefrontal cortex is related to the mechanisms of temporal memory and attention

  12. The Affective Turn

    DEFF Research Database (Denmark)

    Carnera, Alexander

    2012-01-01

    This paper confronts biopolitics with modern labour addressing questions of ‘governmentality’, ‘self-management’ and ‘social innovation’. It argues that the new modes of production within immaterial labour involve a new complex relation between on the one hand the ‘Art of Governance...... of biopolitics that surpasses that of governmentality. The affective self-relation is used as a research tool to analyse the creation of social and economic values in our new modes of productions, for instance, within free labour of the cultural industry. The movie The Five Obstructions is used to show how...... organizing good affective encounters based on limitations enhance and facilitate the performative dimension of self-management. Finally, the paper addresses the problem of critique confronting self-relation with Spinoza's ethics as an ethical difference of powe...

  13. Cytoplasm Affects Embryonic Development

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ Recent studies by CAS researchers furnish strong evidence that a fertilized egg's nucleus isn't the sole site of control for an embryo's development. A research team headed by Prof. Zhu Zuoyan from the CAS Institute of Hydrobiology in Wuhan discovered that cytoplasm affects the number of vertebrae in cloned offspring created when nuclei from one fish genus were transplanted to enucleated eggs of another.

  14. Affective Image Colorization

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hui Wang; Jia Jia; Han-Yu Liao; Lian-Hong Cai

    2012-01-01

    Colorization of gray-scale images has attracted many attentions for a long time.An important role of image color is the conveyer of emotions (through color themes).The colorization with an undesired color theme is less useful,even it is semantically correct.However this has been rarely considered.Automatic colorization respecting both the semantics and the emotions is undoubtedly a challenge.In this paper,we propose a complete system for affective image colorization.We only need the user to assist object segmentation along with text labels and an affective word.First,the text labels along with other object characters are jointly used to filter the internet images to give each object a set of semantically correct reference images.Second,we select a set of color themes according to the affective word based on art theories.With these themes,a generic algorithm is used to select the best reference for each object,balancing various requirements.Finally,we propose a hybrid texture synthesis approach for colorization.To the best of our knowledge,it is the first system which is able to efficiently colorize a gray-scale image semantically by an emotionally controllable fashion.Our experiments show the effectiveness of our system,especially the benefit compared with the previous Markov random field (MRF) based method.

  15. The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends

    DEFF Research Database (Denmark)

    Weterings, Eric; Verkaik, Nicole S; Keijzers, Guido;

    2008-01-01

    Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) an......Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA......-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation...... of DNA-PK(CS) at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK(CS) autophosphorylation at Thr2647 was diminished, while Ser2056...

  16. The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

    Science.gov (United States)

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

  17. A Sir2-like protein participates in mycobacterial NHEJ.

    Directory of Open Access Journals (Sweden)

    Zhongdao Li

    Full Text Available In eukaryotic cells, repair of DNA double-strand breaks (DSBs by the nonhomologous end-joining (NHEJ pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD, whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP screening, we have identified an NAD-dependent deacetylase in mycobacteria which is a homologue of the eukaryotic Sir2 protein and interacts directly with Ku. Results from an in vitro glutathione S-transferase (GST pull-down assay suggest that Sir2 interacts directly with LigD. Plasmid-based end-joining assays revealed that the efficiency of DSB repair in a sir2 deletion mutant was reduced 2-fold. Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR in the stationary phase than the wild-type. Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria.

  18. Interdependence of the Rad50 hook and globular domain functions

    Science.gov (United States)

    Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

    2015-01-01

    SUMMARY Rad50 contains a conserved Zn2+ coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here we focused on rad50 mutations flanking the Zn2+-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end-joining, and DNA double strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled coil and globular ATPase domain, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. PMID:25601756

  19. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Ian Hare

    2016-01-01

    Full Text Available Mesenchymal stem cells (MSCs are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs, the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16 at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

  20. Molecular cloning and analysis of breakpoints on ring chromosome 17 in a patient with autism.

    Science.gov (United States)

    Vazna, Alzbeta; Havlovicova, Marketa; Sedlacek, Zdenek

    2008-01-15

    The breakpoint junction on a ring chromosome 17 in a girl with autism, mental retardation, mild dysmorphism and neurofibromatosis was identified and analysed at the nucleotide level. The extent of the deleted segments was about 1.9 Mb on 17p and about 1.0 Mb on 17q. The structure of the junction between the 17p and 17q arms, especially the lack of significant homology between the juxtaposed genomic regions and the presence of short microhomology at the junction site, indicated non-homologous end joining as the most likely mechanism leading to the rearrangement. In addition to the 17p-17q junction itself, a de novo 1 kb deletion in a distance of 400 bp from the junction was identified, which arose most likely as a part of the rearrangement. The defect directly inactivated 3 genes, and the deleted terminal chromosome segments harboured 27 and 14 protein-coding genes from 17p and 17q, respectively. Several of the genes affected by the rearrangement are candidates for the symptoms observed in the patient. Additional rearrangements similar to the 1 kb deletion observed in our patient may remain undetected but can participate in the phenotype of patients with chromosomal aberrations. They can also be the reason for repeated failures to clone breakpoint junctions in other patients described in the literature.

  1. Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity

    Science.gov (United States)

    Lobachevsky, Pavel; Woodbine, Lisa; Hsiao, Kuang-Chih; Choo, Sharon; Fraser, Chris; Gray, Paul; Smith, Jai; Best, Nickala; Munforte, Laura; Korneeva, Elena; Martin, Roger F.; Jeggo, Penny A.; Martin, Olga A.

    2016-01-01

    Pediatric patients with severe or nonsevere combined immunodeficiency have increased susceptibility to severe, life-threatening infections and, without hematopoietic stem cell transplantation, may fail to thrive. A subset of these patients have the radiosensitive (RS) phenotype, which may necessitate conditioning before hematopoietic stem cell transplantation, and this conditioning includes radiomimetic drugs, which may significantly affect treatment response. To provide statistical criteria for classifying cellular response to ionizing radiation as the measure of functional RS screening, we analyzed the repair capacity and survival of ex vivo irradiated primary skin fibroblasts from five dysmorphic and/or developmentally delayed pediatric patients with severe combined immunodeficiency and combined immunodeficiency. We developed a mathematical framework for the analysis of γ histone 2A isoform X foci kinetics to quantitate DNA-repair capacity, thus establishing crucial criteria for identifying RS. The results, presented in a diagram showing each patient as a point in a 2D RS map, were in agreement with findings from the assessment of cellular RS by clonogenic survival and from the genetic analysis of factors involved in the nonhomologous end-joining repair pathway. We provide recommendations for incorporating into clinical practice the functional assays and genetic analysis used for establishing RS status before conditioning. This knowledge would enable the selection of the most appropriate treatment regimen, reducing the risk for severe therapy-related adverse effects. PMID:26151233

  2. Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity.

    Science.gov (United States)

    Lobachevsky, Pavel; Woodbine, Lisa; Hsiao, Kuang-Chih; Choo, Sharon; Fraser, Chris; Gray, Paul; Smith, Jai; Best, Nickala; Munforte, Laura; Korneeva, Elena; Martin, Roger F; Jeggo, Penny A; Martin, Olga A

    2015-09-01

    Pediatric patients with severe or nonsevere combined immunodeficiency have increased susceptibility to severe, life-threatening infections and, without hematopoietic stem cell transplantation, may fail to thrive. A subset of these patients have the radiosensitive (RS) phenotype, which may necessitate conditioning before hematopoietic stem cell transplantation, and this conditioning includes radiomimetic drugs, which may significantly affect treatment response. To provide statistical criteria for classifying cellular response to ionizing radiation as the measure of functional RS screening, we analyzed the repair capacity and survival of ex vivo irradiated primary skin fibroblasts from five dysmorphic and/or developmentally delayed pediatric patients with severe combined immunodeficiency and combined immunodeficiency. We developed a mathematical framework for the analysis of γ histone 2A isoform X foci kinetics to quantitate DNA-repair capacity, thus establishing crucial criteria for identifying RS. The results, presented in a diagram showing each patient as a point in a 2D RS map, were in agreement with findings from the assessment of cellular RS by clonogenic survival and from the genetic analysis of factors involved in the nonhomologous end-joining repair pathway. We provide recommendations for incorporating into clinical practice the functional assays and genetic analysis used for establishing RS status before conditioning. This knowledge would enable the selection of the most appropriate treatment regimen, reducing the risk for severe therapy-related adverse effects.

  3. Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness

    DEFF Research Database (Denmark)

    Kari, Vijayalakshmi; Mansour, Wael Yassin; Raul, Sanjay Kumar;

    2016-01-01

    The CHD1 gene, encoding the chromo-domain helicase DNA-binding protein-1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double-strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of Ct......IP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR-mediated DNA repair but not non......-homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may...

  4. Characterization of Brca2-deficient plants excludes the role of NHEJ and SSA in the meiotic chromosomal defect phenotype.

    Directory of Open Access Journals (Sweden)

    Marilyn Dumont

    Full Text Available In somatic cells, three major pathways are involved in the repair of DNA double-strand breaks (DBS: Non-Homologous End Joining (NHEJ, Single-Strand Annealing (SSA and Homologous Recombination (HR. In somatic and meiotic HR, DNA DSB are 5' to 3' resected, producing long 3' single-stranded DNA extensions. Brca2 is essential to load the Rad51 recombinase onto these 3' overhangs. The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information. In Arabidopsis, the inactivation of Brca2 specifically during meiosis by an RNAi approach results in aberrant chromosome aggregates, chromosomal fragmentation and missegregation leading to a sterility phenotype. We had previously suggested that such chromosomal behaviour could be due to NHEJ. In this study, we show that knock-out plants affected in both BRCA2 genes show the same meiotic phenotype as the RNAi-inactivated plants. Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants. The role of the plant-specific DNA Ligase6 is also excluded. The possible mechanism(s involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.

  5. Molecular targets in cellular response to ionizing radiation and implications in space radiation protection

    Energy Technology Data Exchange (ETDEWEB)

    Belli, M.; Tabocchini, M.A. [Istituto Superiore di Sanita, Rome (Italy). Physics Lab.; Sapora, O. [Istituto Superiore di Sanita, Rome (Italy). Comparative Toxicology Lab.

    2002-12-01

    DNA repair systems and cell cycle checkpoints closely co-operate in the attempt of maintaining the genomic integrity of cells damaged by ionizing radiation. DNA double-strand breaks (DSB) are considered as the most biologically important radiation-induced damage. Their spatial distribution and association with other types of damage depend on radiation quality. It is believed these features affect damage reparability, thus explaining the higher efficiency for cellular effects of densely ionizing radiation with respect to {gamma}-rays. DSB repair systems identified in mammalian cells are homologous recombination (HR), single-strand annealing (SSA) and non-homologous end-joining (NHEJ). Some enzymes may participate in more than one of these repair systems. DNA damage also triggers biochemical signals activating checkpoints responsible for delay in cell cycle progression that allows more time for repair. Those at G1/S and S phases prevent replication of damaged DNA and those at G2/M phase prevent segregation of changed chromosomes. Individuals with lack or alterations of genes involved in DNA DSB repair and cell cycle checkpoints exhibit syndromes characterized by genome instability and predisposition to cancer. Information reviewed in this paper on the basic mechanisms of cellular response to ionizing radiation indicates their importance for a number of issues relevant to protection of astronauts from space radiation. (author)

  6. Roles of XRCC2, RAD51B and RAD51D in RAD51-independent SSA recombination.

    Directory of Open Access Journals (Sweden)

    Heïdi Serra

    2013-11-01

    Full Text Available The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.

  7. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    Science.gov (United States)

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.

  8. DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair.

    Science.gov (United States)

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.

  9. DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Aya Kurosawa

    Full Text Available Nonhomologous end-joining (NHEJ and homologous recombination (HR are two major pathways for repairing DNA double-strand breaks (DSBs; however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.

  10. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry.

    Science.gov (United States)

    Lee, Kyung-Jong; Shang, Zeng-Fu; Lin, Yu-Fen; Sun, Jingxin; Morotomi-Yano, Keiko; Saha, Debabrata; Chen, Benjamin P C

    2015-04-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs-deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

  11. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry

    Directory of Open Access Journals (Sweden)

    Kyung-Jong Lee

    2015-04-01

    Full Text Available DNA-dependent protein kinase catalytic subunit (DNA-PKcs is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1, the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1, which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs–deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

  12. Genome-Wide Mapping of Structural Variations Reveals a Copy Number Variant That Determines Reproductive Morphology in Cucumber.

    Science.gov (United States)

    Zhang, Zhonghua; Mao, Linyong; Chen, Huiming; Bu, Fengjiao; Li, Guangcun; Sun, Jinjing; Li, Shuai; Sun, Honghe; Jiao, Chen; Blakely, Rachel; Pan, Junsong; Cai, Run; Luo, Ruibang; Van de Peer, Yves; Jacobsen, Evert; Fei, Zhangjun; Huang, Sanwen

    2015-06-01

    Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.

  13. Microhomology-mediated deletion and gene conversion in African trypanosomes.

    Science.gov (United States)

    Glover, Lucy; Jun, Junho; Horn, David

    2011-03-01

    Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.

  14. Risk, Affect and Emotion

    Directory of Open Access Journals (Sweden)

    Jens O. Zinn

    2006-01-01

    Full Text Available For a long time theorising has underestimated the importance of affect and emotion in decision making and the management of risk and uncertainty. In relatively one-sided interpretations emotions were often interpreted as threats for rational decision making, and could be triggered by uncertainties, which would go along with social change. Recent interdisciplinary research has shown the importance to acknowledge the more complex link between reasoning and emotions. The article outlines different perspectives on emotion in risk research of economics, psychology and sociology and argues for further research. URN: urn:nbn:de:0114-fqs0601293

  15. Visual affect recognition

    CERN Document Server

    Stathopoulou, I-O

    2010-01-01

    It is generally known that human faces, as well as body motions and gestures, provide a wealth of information about a person, such as age, race, sex and emotional state. This monograph primarily studies the perception of facial expression of emotion, and secondarily of motion and gestures, with the purpose of developing a fully automated visual affect recognition system for use in modes of human/computer interaction. The book begins with a survey of the literature on emotion perception, followed by a description of empirical studies conducted with human participants and the construction of a '

  16. Multisensory Perception of Affect

    Directory of Open Access Journals (Sweden)

    Beatrice de Gelder

    2011-10-01

    Full Text Available Multisensory integration must stand out among the fields of research that have witnessed a most impressive explosion of interest this last decade. One of these new areas of multisensory research concerns emotion. Since our first exploration of this phenomenon (de Gelder et al., 1999 a number of studies have appeared and they have used a wide variety of behavioral, neuropsychological and neuroscientifc methods. The goal of this presentation is threefold. First, we review the research on audiovisual perception of emotional signals from the face and the voice followed by a report or more recent studies on integrating emotional information provided by the voice and whole body expressions. We will also include some recent work on multisensory music perception. In the next section we discuss some methodological and theoretical issues. Finally, we will discuss findings about abnormal affective audiovisual integration in schizophrenia and in autism.

  17. Trauma-affected refugees

    DEFF Research Database (Denmark)

    Sonne, Charlotte Kærgaard

    2016-01-01

    . The aim of PTF3 was to examine differences in the effects of venlafaxine and sertraline on Post-traumatic Stress Disorder (PTSD), depression and functional impairments in trauma-affected refugees as well as research predictors for treatment outcome. The patients included were 207 adult refugees diagnosed......-reported depression and anxiety symptoms measured on Hopkins Symptom Check List-25 (HSCL-25), self-reported social functioning measured on the Social Adjustment Scale Self Report, short version (SAS-SR), and observer-rated depression and anxiety symptoms assessed on the Hamilton Depression and Anxiety Ratings Scales......=0.06). The only item from the rating scale that was significantly correlated to outcome on HTQ was job status, while a number of other items were significantly related to changes in depression and anxiety symptoms. The size of correlation coefficients was, however, modest. In addition, we found...

  18. Security affects us all!

    CERN Multimedia

    SMB Department

    2016-01-01

    In the hope of minimising the number of thefts of the Organization’s property, which can lead to months of work going to waste on certain projects, you are reminded of the importance that CERN attaches to the rules concerning the protection of equipment for which we are responsible. If you see any unusual behaviour or if you are the victim of a theft, don’t hesitate to report it by submitting a ticket through the CERN Portal or calling the CSA. Security affects us all!   CERN is attractive in more ways than one, and it remains as attractive as ever to thieves. With the nice weather and with the holiday season in full swing, the number of thefts recorded at CERN is on the rise. Items stolen include money, computers, electronic equipment, cable drums and copper antennae.   There are a few basic precautions that you should take to protect both your own and the Organization’s property: lock your door, don’t leave valuable items in your office, st...

  19. Encountering Science Education's Capacity to Affect and Be Affected

    Science.gov (United States)

    Alsop, Steve

    2016-01-01

    What might science education learn from the recent affective turn in the humanities and social sciences? Framed as a response to Michalinos Zembylas's article, this essay draws from selected theorizing in affect theory, science education and science and technology studies, in pursuit of diverse and productive ways to talk of affect within science…

  20. Mechanistic Modelling and Bayesian Inference Elucidates the Variable Dynamics of Double-Strand Break Repair

    Science.gov (United States)

    2016-01-01

    DNA double-strand breaks are lesions that form during metabolism, DNA replication and exposure to mutagens. When a double-strand break occurs one of a number of repair mechanisms is recruited, all of which have differing propensities for mutational events. Despite DNA repair being of crucial importance, the relative contribution of these mechanisms and their regulatory interactions remain to be fully elucidated. Understanding these mutational processes will have a profound impact on our knowledge of genomic instability, with implications across health, disease and evolution. Here we present a new method to model the combined activation of non-homologous end joining, single strand annealing and alternative end joining, following exposure to ionising radiation. We use Bayesian statistics to integrate eight biological data sets of double-strand break repair curves under varying genetic knockouts and confirm that our model is predictive by re-simulating and comparing to additional data. Analysis of the model suggests that there are at least three disjoint modes of repair, which we assign as fast, slow and intermediate. Our results show that when multiple data sets are combined, the rate for intermediate repair is variable amongst genetic knockouts. Further analysis suggests that the ratio between slow and intermediate repair depends on the presence or absence of DNA-PKcs and Ku70, which implies that non-homologous end joining and alternative end joining are not independent. Finally, we consider the proportion of double-strand breaks within each mechanism as a time series and predict activity as a function of repair rate. We outline how our insights can be directly tested using imaging and sequencing techniques and conclude that there is evidence of variable dynamics in alternative repair pathways. Our approach is an important step towards providing a unifying theoretical framework for the dynamics of DNA repair processes. PMID:27741226

  1. Drugs affecting the eye.

    Science.gov (United States)

    Taylor, F

    1985-08-01

    This discussion reviews drugs that affect the eye, including antihyperglycemic agents; corticosteroids; antirheumatic drugs (quinolines, indomethacin, and allopurinol); psychiatric drugs (phenothiazine, thioridazine, and chlorpromazine); drugs used in cardiology (practolol, amiodarone, and digitalis gylcosides); drugs implicated in optic neuritis and atrophy, drugs with an anticholinergic action; oral contraceptives (OCs); and topical drugs and systemic effects. Refractive changes, either myopic or hypermetropic, can occur as a result of hyperglycemia, and variation in vision is sometimes a presenting symptom in diabetes mellitus. If it causes a change in the refraction, treatment of hyperglycemia almost always produces a temporary hypermetropia. A return to the original refractive state often takes weeks, sometimes months. There is some evidence that patients adequately treated with insulin improve more rapidly than those taking oral medication. Such patients always should be referred for opthalmological evaluation as other factors might be responsible, but it might not be possible to order the appropriate spectacle correction for some time. The most important ocular side effect of the systemic adiministration of corticosteroids is the formation of a posterior subcapsular cataract. Glaucoma also can result from corticosteroids, most often when they are applied topically. Corticosteroids have been implicated in the production of benign intracranial hypertension, which is paradoxical because they also are used in its treatment. The most important side effect of drugs such as chloroquine and hydroxychloroquine is an almost always irreversible maculopathy with resultant loss of central vision. Corneal and retinal changes similar to those caused by the quinolines have been reported with indomethacin, but there is some question about a cause and effect relationship. The National Registry of Drug Induced Ocular Side Effects in the US published 30 case histories of

  2. Affective monitoring: A generic mechanism for affect elicitation

    Directory of Open Access Journals (Sweden)

    Hans ePhaf

    2012-03-01

    Full Text Available In this paper we sketch a new framework for affect elicitation, which is based on previous evolutionary and connectionist modeling and experimental work from our group. Affective monitoring is considered a local match-mismatch process within a module of the neural network. Negative affect is raised instantly by mismatches, incongruency, disfluency, novelty, incoherence, and dissonance, whereas positive affect follows from matches, congruency, fluency, familiarity, coherence, and resonance, at least when an initial mismatch can be solved quickly. Affective monitoring is considered an evolutionary-early conflict and change detection process operating at the same level as, for instance, attentional selection. It runs in parallel and imparts affective flavour to emotional behavior systems, which involve evolutionary-prepared stimuli and action tendencies related to for instance defensive, exploratory, attachment, or appetitive behavior. Positive affect is represented in the networks by high-frequency oscillations, presumably in the gamma band. Negative affect corresponds to more incoherent lower-frequency oscillations, presumably in the theta band. For affect to become conscious, large-scale synchronization of the oscillations over the network and the construction of emotional experiences are required. These constructions involve perceptions of bodily states and action tendencies, but also appraisals as well as efforts to regulate the emotion. Importantly, affective monitoring accompanies every kind of information processing, but conscious emotions, which result from the later integration of affect in a cognitive context, are much rarer events.

  3. Affective Productions of Mathematical Experience

    Science.gov (United States)

    Walshaw, Margaret; Brown, Tony

    2012-01-01

    In underscoring the affective elements of mathematics experience, we work with contemporary readings of the work of Spinoza on the politics of affect, to understand what is included in the cognitive repertoire of the Subject. We draw on those resources to tell a pedagogical tale about the relation between cognition and affect in settings of…

  4. Application of Affect to CALL

    Institute of Scientific and Technical Information of China (English)

    李露

    2006-01-01

    This essay mainly attempts to discuss some affective factors in CALL, with focus on affective influence on English (foreign language) learning and thus concludes that in the process of CALL related affect should be activated and made good use of to better foreign language teaching and learning.

  5. Social and Affective Robotics Tutorial

    NARCIS (Netherlands)

    Pantic, Maja; Evers, Vanessa; Deisenroth, Marc; Merino, Luis; Schuller, Björn

    2016-01-01

    Social and Affective Robotics is a growing multidisciplinary field encompassing computer science, engineering, psychology, education, and many other disciplines. It explores how social and affective factors influence interactions between humans and robots, and how affect and social signals can be se

  6. Affective Induction and Creative Thinking

    Science.gov (United States)

    Fernández-Abascal, Enrique G.; Díaz, María D. Martín

    2013-01-01

    Three studies explored the relation between affect and production of creative divergent thinking, assessed with the Torrance Tests of Creative Thinking (Figural TTCT). In the first study, general, positive, and negative affect, assessed with the Positive and Negative Affect Scale (PANAS) were compared with creative production. In the second study,…

  7. Assistive Technology and Affective Mediation

    Directory of Open Access Journals (Sweden)

    Nestor Garay

    2006-01-01

    Full Text Available The lack of attention towards affective communication in assistive technology and disability research can be potentially overcome thanks to the development of affective computing and affective mediation areas. This document describes the main impairments and disorders that may involve affective communication deficits. We also present several affective mediation technologies that are being applied or may be integrated in assistive technologies in order to improve affective communication for a range of disabilities. Finally, we describe our experience with Gestele, an application that incorporates multimodal elements of affective mediation for people with mobility impairments, as well as the results of an empirical study conducted to evaluate the application’s validity in communication via telephone.

  8. Encountering science education's capacity to affect and be affected

    Science.gov (United States)

    Alsop, Steve

    2016-09-01

    What might science education learn from the recent affective turn in the humanities and social sciences? Framed as a response to Michalinos Zembylas's article, this essay draws from selected theorizing in affect theory, science education and science and technology studies, in pursuit of diverse and productive ways to talk of affect within science education. These discussions are framed by desires to transcend traditional epistemic boundaries and practices. The article concludes offering some associated ambiguities and tensions involved.

  9. DNA-PKcs Expression Predicts Response to Radiotherapy in Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bouchaert, Patrick [Department of Pathology, CHU-Universite de Poitiers, Poitiers (France); Department of Medical Oncology, CHU-Universite de Poitiers, Poitiers (France); Guerif, Stephane [Department of Radiation Oncology, CHU-Universite de Poitiers, Poitiers (France); Debiais, Celine [Department of Pathology, CHU-Universite de Poitiers, Poitiers (France); Irani, Jacques [Department of Urology, CHU-Universite de Poitiers, Poitiers (France); Fromont, Gaelle, E-mail: g.fromont@chu-poitiers.fr [Department of Pathology, CHU-Universite de Poitiers, Poitiers (France)

    2012-12-01

    Purpose: Double-strand breaks, the most lethal DNA lesions induced by ionizing radiation, are mainly repaired by the nonhomologous end-joining system. The expression of the nonhomologous end-joining pathway has never been studied in prostate cancer, and its prognostic value for patients undergoing radiotherapy remains unknown. Methods: Pretreatment biopsies from 238 patients treated with exclusive external beam radiotherapy for localized prostate cancer with {>=}2 years of follow-up were reviewed to reassess the Gleason score. Of these 238 cases, 179 were suitable for in situ analysis and were included in the tissue microarrays. Expression of the nonhomologous end-joining proteins Ku70, Ku80, DNA-dependent protein kinase, catalytic subunits (DNA-PKcs), and X-ray repair cross complementing 4-like factor was studied by immunohistochemistry, together with the proliferation marker Ki67. Results: The predictive value of the Gleason score for biochemical relapse (using the Phoenix criteria) was markedly improved after review (P<.0001) compared with the initial score (P=.003). The clinical stage, pretreatment prostate-specific antigen level, and perineural invasion status were also associated with progression-free survival (P=.005, P<.0001, and P=.03, respectively). High proliferation (>4%) tends to be associated with biochemical recurrence; however, the difference did not reach statistical significance (P=.06). Although the expression of Ku70, Ku80, and X-ray repair cross complementing 4-like factor was not predictive of relapse, positive DNA-PKcs nuclear staining was closely associated with biochemical recurrence (P=.0002). On multivariate analysis, only the Gleason score, prostate-specific antigen level, and DNA-PKcs status remained predictive of recurrence (P=.003, P=.002, and P=.01, respectively). Conclusions: The results of the present study highly suggest that DNA-PKcs could be a predictive marker of recurrence after radiotherapy, independently of the classic

  10. Cellulases: ambiguous nonhomologous enzymes in a genomic perspective.

    Science.gov (United States)

    Sukharnikov, Leonid O; Cantwell, Brian J; Podar, Mircea; Zhulin, Igor B

    2011-10-01

    The key material for bioethanol production is cellulose, which is one of the main components of the plant cell wall. Enzymatic depolymerization of cellulose is an essential step in bioethanol production, and can be accomplished by fungal and bacterial cellulases. Most of the biochemically characterized bacterial cellulases come from only a few cellulose-degrading bacteria, thus limiting our knowledge of a range of cellulolytic activities that exist in nature. The recent explosion of genomic data offers a unique opportunity to search for novel cellulolytic activities; however, the absence of clear understanding of structural and functional features that are important for reliable computational identification of cellulases precludes their exploration in the genomic datasets. Here, we explore the diversity of cellulases and propose a genomic approach to overcome this bottleneck.

  11. The Non-Homologous Nature of Solar Diameter Variations

    CERN Document Server

    Sofia, S; Demarque, P; Li, L; Thuillier, G; Sofia, Sabatino; Basu, Sarbani; Demarque, Pierre; Li, Linghuai; Thuillier, Gerard

    2005-01-01

    We show in this paper that the changes of the solar diameter in response to variations of large scale magnetic fields and turbulence are not homologous. For the best current model, the variation at the photospheric level is over 1000 times larger than the variation at a depth of 5 Mm, which is about the level at which f-mode solar oscillations determine diameter variations. This model is supported by observations that indicate larger diameter changes for high degree f-modes than for low degree f-modes, since energy of the former are concentrated at shallower layers than the latter.

  12. Current Progress in Therapeutic Gene Editing for Monogenic Diseases.

    Science.gov (United States)

    Prakash, Versha; Moore, Marc; Yáñez-Muñoz, Rafael J

    2016-03-01

    Programmable nucleases allow defined alterations in the genome with ease-of-use, efficiency, and specificity. Their availability has led to accurate and widespread genome engineering, with multiple applications in basic research, biotechnology, and therapy. With regard to human gene therapy, nuclease-based gene editing has facilitated development of a broad range of therapeutic strategies based on both nonhomologous end joining and homology-dependent repair. This review discusses current progress in nuclease-based therapeutic applications for a subset of inherited monogenic diseases including cystic fibrosis, Duchenne muscular dystrophy, diseases of the bone marrow, and hemophilia and highlights associated challenges and future prospects.

  13. Fast and sensitive detection of indels induced by precise gene targeting

    DEFF Research Database (Denmark)

    Yang, Zhang; Steentoft, Catharina; Hauge, Camilla

    2015-01-01

    The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect...... and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect...

  14. DNA end resection: many nucleases make light work

    OpenAIRE

    Mimitou, Eleni P.; Symington, Lorraine S.

    2009-01-01

    Double strand breaks (DSBs) are deleterious DNA lesions and if left unrepaired result in severe genomic instability. Cells use two main pathways to repair DSBs: homologous recombination (HR) or non-homologous end joining (NHEJ) depending on the phase of the cell cycle and the nature of the DSB ends. A key step where pathway choice is exerted is in the ‘licensing’ of 5′-3′ resection of the ends to produce recombinogenic 3′ single-stranded tails. These tails are substrate for binding by Rad51 t...

  15. The HTLV-1 Tax Oncoprotein Represses Ku80 Gene Expression

    OpenAIRE

    Ducu, Razvan I.; Dayaram, Tajhal; Marriott, Susan J

    2011-01-01

    The HTLV-I oncoprotein Tax interferes with DNA double strand break repair. Since non-homologous end joining (NHEJ) is a major pathway used to repair DNA double strand breaks we examined the effect of Tax on this pathway, with particular interest in the expression and function of Ku80, a critical component of the NHEJ pathway. Tax expression decreased Ku80 mRNA and protein levels, and repressed transcription from the Ku80 promoter. Conversely, Ku80 mRNA increased following siRNA knockdown of T...

  16. Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

    OpenAIRE

    Yang, Yun-Gui; Saidi, Amal; Frappart, Pierre-Olivier; Min, WooKee; Barrucand, Christelle; Dumon-Jones, Valérie; Michelon, Jocelyne; Herceg, Zdenko; Wang, Zhao-Qi

    2006-01-01

    NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting...

  17. Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs).

    Science.gov (United States)

    Taheri-Ghahfarokhi, Amir; Malaver-Ortega, Luis F; Sumer, Huseyin

    2015-01-01

    Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.

  18. CRISPR-Cas9-guided Genome Engineering in C. elegans

    Science.gov (United States)

    Kim, Hyun-Min; Colaiácovo, Monica P.

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms including the nematode C. elegans. Recent studies developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning and injection methods required for delivering Cas9, sgRNAs and repair template DNA into the C. elegans germline. PMID:27366893

  19. Caracterización estructural mediante microscopía electrónica del complejo Xrcc4-Dna Ligasa IV, implicado en reparación de roturas del DNA

    OpenAIRE

    Recuero-Checa, María Ángeles

    2010-01-01

    Las células de cualquier organismo vivo están sometidas a multitud de amenazas tanto exógenas como endógenas que pueden dañar el genoma. Existen diversos tipos de roturas en el DNA, siendo las roturas de doble cadena unas de las más peligrosas para l a supervivencia celular. En eucariotas superiores, estas roturas son reparadas principalmente mediante el mecanismo de reparación de unión de extremos no homólogos (también llamado NHEJ, del inglés Non-homologous end-joining), donde los extremos ...

  20. Genome engineering with TALENs and ZFNs: repair pathways and donor design.

    Science.gov (United States)

    Carroll, Dana; Beumer, Kelly J

    2014-09-01

    Genome engineering with targetable nucleases depends on cellular pathways of DNA repair after target cleavage. Knowledge of how those pathways work, their requirements and their active factors, can guide experimental design and improve outcomes. While many aspects of both homologous recombination (HR) and nonhomologous end joining (NHEJ) are shared by a broad range of cells and organisms, some features are specific to individual situations. This article reviews the influence of repair mechanisms on the results of gene targeting experiments, with an emphasis on lessons learned from experiments with Drosophila.

  1. CRISPR/Cas9 mediated genome engineering in Drosophila.

    Science.gov (United States)

    Bassett, Andrew; Liu, Ji-Long

    2014-09-01

    Genome engineering has revolutionised genetic analysis in many organisms. Here we describe a simple and efficient technique to generate and detect novel mutations in desired target genes in Drosophila melanogaster. We target double strand breaks to specific sites within the genome by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed synthetic guide RNA into Drosophila embryos. The small insertion and deletion mutations that result from inefficient non-homologous end joining at this site are detected by high resolution melt analysis of whole flies and individual wings, allowing stable lines to be made within 1 month.

  2. SCAI promotes DNA double-strand break repair in distinct chromosomal contexts

    DEFF Research Database (Denmark)

    Hansen, Rebecca Kring; Mund, Andreas; Poulsen, Sara Lund

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer...... in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI...

  3. E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

    OpenAIRE

    2008-01-01

    The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC ...

  4. Seasonal Affective Disorder: For Teens

    Science.gov (United States)

    ... can affect concentration, too, interfering with a person's school performance and grades. A student may have more trouble ... less motivated or is making less effort in school. Less time ... can affect self-esteem and leave a person feeling disappointed, isolated, and ...

  5. Flow, affect and visual creativity.

    Science.gov (United States)

    Cseh, Genevieve M; Phillips, Louise H; Pearson, David G

    2015-01-01

    Flow (being in the zone) is purported to have positive consequences in terms of affect and performance; however, there is no empirical evidence about these links in visual creativity. Positive affect often--but inconsistently--facilitates creativity, and both may be linked to experiencing flow. This study aimed to determine relationships between these variables within visual creativity. Participants performed the creative mental synthesis task to simulate the creative process. Affect change (pre- vs. post-task) and flow were measured via questionnaires. The creativity of synthesis drawings was rated objectively and subjectively by judges. Findings empirically demonstrate that flow is related to affect improvement during visual creativity. Affect change was linked to productivity and self-rated creativity, but no other objective or subjective performance measures. Flow was unrelated to all external performance measures but was highly correlated with self-rated creativity; flow may therefore motivate perseverance towards eventual excellence rather than provide direct cognitive enhancement.

  6. 组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用%Effect of Histone Deacetylase Inhibitors on DNA DSB Repair

    Institute of Scientific and Technical Information of China (English)

    孙有湘; 周克元; 李莉萍

    2013-01-01

    DNA双链断裂(double strand breaks,DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶(histone deacetylase)作为抗肿瘤治疗的新靶标,其抑制剂(histone deacetylase inhibitors,HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.%DNA double strand break (DSB) can be fatal to cells.The non-homologous end joining (NHEJ),homologous direct recombination (HDR),single strand annealing (SSA) and micro-homology mediated end joining (MMEJ) pathways are able to repair DSBs in vivo.A main strategy of clinical radiotherapy and chemotherapy is to induce chromosomal DNA damage that are difficult to repair,thus to cause cell cycle arrest,apoptosis or necrosis in tumor cells.As a new target of anti-tumor medicine,histonc deacetylase inhibitor (HDACi) effectively suppresses DSBs repair capacity and sensitize tumor cells to radiotherapy or chemotherapy.Recent research suggested that the error-free DSBs repair pathways HDR and classical-NHEJ were suppressed by HDACi in tumor cells,and the error-prone SSA and MMEJ pathways might also involved in.Although much progress have acquired on the mechanism of how HDACi affecting DSB repair pathways,there are still many questions need to be elucidated.

  7. Affective Computing and Intelligent Interaction

    CERN Document Server

    2012-01-01

    2012 International Conference on Affective Computing and Intelligent Interaction (ICACII 2012) was the most comprehensive conference focused on the various aspects of advances in Affective Computing and Intelligent Interaction. The conference provided a rare opportunity to bring together worldwide academic researchers and practitioners for exchanging the latest developments and applications in this field such as Intelligent Computing, Affective Computing, Machine Learning, Business Intelligence and HCI.   This volume is a collection of 119 papers selected from 410 submissions from universities and industries all over the world, based on their quality and relevancy to the conference. All of the papers have been peer-reviewed by selected experts.  

  8. The affective turn in ethnomusicology

    Directory of Open Access Journals (Sweden)

    Hofman Ana

    2015-01-01

    Full Text Available The affective turn, which has already questioned dominant paradigms in many disciplinary fields including cultural studies, philosophy, political theory, anthropology, psychology and neuroscience, has started to attract more attention in the field of ethnomusicology, becoming a particularly vibrant stream of thought. Drawing on the voices that call for the historicisation of and critical deliberation on the field of affect studies, the article strives to show how theories of affect might expand dominant paradigms in ethnomusicology and also points to their limitations.

  9. Stress factors in affective diseases.

    Science.gov (United States)

    Bidzińska, E J

    1984-02-01

    An investigation carried out on 97 patients with affective disorders and on 100 healthy control subjects, revealed that acute and chronic stress factors occurred more in the group of patients with affective disorders than among healthy control over a similar time period. The frequency of stressful life situations was the same before the first affective episode in patients with unipolar and bipolar illness. The possible participation of such factors in triggering the first phase of illness is discussed. Similar factors appeared in both types of affective disorders. Significantly more frequent among patients than in the control group were: marital and family conflicts, health problems, emotional and ambitional failures, lack of success and work overload.

  10. A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

    Directory of Open Access Journals (Sweden)

    Changrim Lee

    Full Text Available PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs, while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs. In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

  11. [Affective disorders and eating disorders].

    Science.gov (United States)

    Fakra, Eric; Belzeaux, R; Azorin, J M; Adida, M

    2014-12-01

    Epidemiologic studies show a frequent co-occurence of affective and eating disorders. The incidence of one disorder in patients suffering from the other disorder is well over the incidence in the general population. Several causes could explain this increased comorbidity. First, the iatrogenic origin is detailed. Indeed, psychotropic drugs, and particularly mood stabilizers, often lead to modification in eating behaviors, generally inducing weight gain. These drugs can increase desire for food, reduce baseline metabolism or decrease motor activity. Also, affective and eating disorders share several characteristics in semiology. These similarities can not only obscure the differential diagnosis but may also attest of conjoint pathophysiological bases in the two conditions. However, genetic and biological findings so far are too sparse to corroborate this last hypothesis. Nonetheless, it is noteworthy that comorbidity of affective and eating disorders worsens patients'prognosis and is associated with more severe forms of affective disorders characterized by an earlier age of onset in the disease, higher number of mood episodes and a higher suicidality. Lastly, psychotropic drugs used in affective disorders (lithium, antiepileptic mood stabilizers, atypical antipsychotics, antidepressants) are reviewed in order to weigh their efficacy in eating disorders. This could help establish the best therapeutic option when confronted to comorbidity.

  12. Staging of unipolar affective illness

    Directory of Open Access Journals (Sweden)

    Ewa Ferensztajn

    2014-12-01

    Full Text Available In this article, a concept of staging of unipolar affective illness (recurrent depression is presented. In respective subchapters, three most important aspects of this issue have been discussed: 1 staging of unipolar affective illness; 2 staging of treatment-resistant depression; and 3 conversion of unipolar into bipolar affective illness. The evidence for so called neuroprogression of the illness, accumulated in recent years, has allowed for a classification of staging based on a concept of allostasis and allostatic load. In the course of illness, changes in neuroendocrine system (mainly hypothalamic pituitary-adrenal (HPA axis, immunological system, mechanisms of oxidative stress, neurotransmitters, neurotrophic factors as well as structural and functional changes of the brain occur. In their paper of 2007, Fava and Tossani elaborated a concept of staging of unipolar affective illness presenting a continuum model of five consecutive stages with specific clinical features. In the present paper, a concept of treatment-resistant depression and staging of treatment resistance is presented in the context of several models. An important determinant of treatment-resistant depression is so called subthreshold bipolarity which is connected with worse efficacy of antidepressant drugs. In the course of illness, there is a possibility of changing diagnosis from recurrent depression into bipolar affective illness. The studies on this issue show that frequency of such diagnostic conversion is 1,5% of depressed patients per year.

  13. On Factors Affecting Listening Comprehension

    Institute of Scientific and Technical Information of China (English)

    苗琴

    2014-01-01

    In English teaching and learning, listening ability is an important part of communicative competence, is a very practical integrated skill. It has been a difficult skill in second language acquisition for many students. Many Chinese students are skilled in reading, but often they tend to neglect the listening. However, owing to the higher requirements of many English tests and the great importance in communication, students begin to pay attention to develop their English listening skills. But there are many factors affecting listening, the paper mainly focuses on linguistic factors and non-linguistic factors that affect listening, to provide a theoretical basis to help exploring ways of improving listening and comprehension skills.

  14. Industrial applications of affective engineering

    CERN Document Server

    Shiizuka, Hisao; Lee, Kun-Pyo; Otani, Tsuyoshi; Lim, Chee-Peng

    2014-01-01

    This book examines the industrial applications of affective engineering. The contributors cover new analytical methods such as fluctuation, fuzzy logic, fractals, and complex systems. These chapters also include interdisciplinary research that traverses a wide range of fields, including information engineering, human engineering, cognitive science, psychology, and design studies. The text is split into two parts: theory and applications. This work is a collection of the best papers from ISAE2013 (International Symposium of Affective Engineering) held at Kitakyushu, Japan and Japan Kansei Engineering Meeting on March 6-8, 2013.

  15. Affect, Emotion and Similar Concepts

    Directory of Open Access Journals (Sweden)

    J.L. Pio Abreu

    2013-12-01

    Full Text Available The author begins by commenting on the difficulty of the semantic delimitation between concepts of affect, emotion, sentiment, feeling, mood, and passion. This difficulty becomes greater when the terms are translated into different languages. He then foccuses on the concept of emotion, which has benefited from recent research, and its distinction from mood, a concept which is at the base of the psychopathology of bipolar disorders. Much more complex is the Portuguese concept of affect (different from the English concept, which has an interpersonal dimension and can be developed from recent discoveries of “mirror neurons” and “theory of mind”.

  16. Trends affecting hospitals' human resources.

    Science.gov (United States)

    Neudeck, M M

    1985-01-01

    Hospital workers at every level--from administrators to housekeepers--will be affected by the interaction of changes already underway in the healthcare industry. Societal forces that will affect the hospital workforce include demographic change, the rise of the participatory ethic and decentralization, a growing philosophy of job entitlement, and new pressures for unionization. At the same time, the industry is faced with changing manpower requirements, cost containment, and the oversupply of physicians. This article identifies some of the likely effects of these changes on hospital human resources and suggests ways that administrators can prepare for them.

  17. Affective Computing and Sentiment Analysis

    CERN Document Server

    Ahmad, Khurshid

    2011-01-01

    This volume maps the watershed areas between two 'holy grails' of computer science: the identification and interpretation of affect -- including sentiment and mood. The expression of sentiment and mood involves the use of metaphors, especially in emotive situations. Affect computing is rooted in hermeneutics, philosophy, political science and sociology, and is now a key area of research in computer science. The 24/7 news sites and blogs facilitate the expression and shaping of opinion locally and globally. Sentiment analysis, based on text and data mining, is being used in the looking at news

  18. Come, see and experience affective interactive art

    NARCIS (Netherlands)

    Bialoskorski, Leticia S.S.; Westerink, Joyce H.D.M.; Broek, van den Egon L.; Nijholt, Anton; Reidsma, Dennis; Hondorp, Hendri

    2009-01-01

    The progress in the field of affective computing enables the realization of affective consumer products, affective games, and affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on

  19. Mood Swings: An Affective Interactive Art System

    NARCIS (Netherlands)

    Bialoskorski, Leticia S.S.; Westerink, Joyce H.D.M.; Broek, van den Egon L.; Nijholt, Anton; Reidsma, Dennis; Hondorp, Hendri

    2009-01-01

    The progress in the field of affective computing enables the realization of affective consumer products, affective games, and affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on

  20. Come, See and Experience Affective Interactive Art

    NARCIS (Netherlands)

    Bialoskorski, Leticia S.S.; Westerink, Joyce H.D.M.; Broek, van den Egon L.; Nijholt, A.; Reidsma, D.; Hondorp, G.H.W.

    2009-01-01

    The progress in the field of affective computing enables the realization of affective consumer products, affective games, and affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on

  1. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.

    Directory of Open Access Journals (Sweden)

    Nicole Bennardo

    2008-06-01

    Full Text Available Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ, single-strand annealing (SSA, and homology directed repair (HDR/GC. Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI-induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

  2. Affecting Critical Thinking through Speech.

    Science.gov (United States)

    O'Keefe, Virginia P.

    Intended for teachers, this booklet shows how spoken language can affect student thinking and presents strategies for teaching critical thinking skills. The first section discusses the theoretical and research bases for promoting critical thinking through speech, defines critical thinking, explores critical thinking as abstract thinking, and tells…

  3. Urban Interaction and Affective Experience

    DEFF Research Database (Denmark)

    Fritsch, Jonas; Brynskov, Martin

    2008-01-01

    in favor of three points in that direction: First we argue that interaction – and the definition of interaction – is central to unfold the potential of digital urban media, from big, shared screens and media facades to small pri-vate, networked mobile and embedded platforms. Then we argue that an affective...

  4. Intuition, affect, and peculiar beliefs

    NARCIS (Netherlands)

    Boden, M.T.; Berenbaum, H.; Topper, M.

    2012-01-01

    Research with college students has found that intuitive thinking (e.g., using hunches to ascribe meaning to experiences) and positive affect interactively predict ideas of reference and odd/magical beliefs. We investigated whether these results would generalize to a diverse community sample of adult

  5. Test Expectancy Affects Metacomprehension Accuracy

    Science.gov (United States)

    Thiede, Keith W.; Wiley, Jennifer; Griffin, Thomas D.

    2011-01-01

    Background: Theory suggests that the accuracy of metacognitive monitoring is affected by the cues used to judge learning. Researchers have improved monitoring accuracy by directing attention to more appropriate cues; however, this is the first study to more directly point students to more appropriate cues using instructions regarding tests and…

  6. Do recruitment ties affect wages?

    DEFF Research Database (Denmark)

    Larsen, Anna Folke; Rand, John; Torm, Nina Elisabeth

    This paper examines the extent to which recruitment ties affect individual wage outcomes in small and medium scale manufacturing firms. Based on a unique matched employer-employee dataset from Vietnam we find that there is a significant positive wage premium associated with obtaining a job through...

  7. Clinical Judgment and Affective Disorders.

    Science.gov (United States)

    Strohmer, Douglas C.; And Others

    1984-01-01

    Addressed the limitations of previous work on counselor clinical judgment in a study involving 20 counselors who were asked to make a series of judgments. Results suggested the judgment processes of experienced counselors making diagnoses of affective disorders differs depending on the type of judgment. (JAC)

  8. How indoor environment affects performance

    DEFF Research Database (Denmark)

    Wyon, David Peter; Wargocki, Pawel

    2013-01-01

    As experienced researchers in the effects of thermal comfort and indoor air quality on performance, we are often asked to give our best estimate of how, and to what extent, performance is affected by different aspects of indoor climate. This article provides a brief summary of our personal opinions...

  9. Aesthetics, Affect, and Educational Politics

    Science.gov (United States)

    Means, Alex

    2011-01-01

    This essay explores aesthetics, affect, and educational politics through the thought of Gilles Deleuze and Jacques Ranciere. It contextualizes and contrasts the theoretical valences of their ethical and democratic projects through their shared critique of Kant. It then puts Ranciere's notion of dissensus to work by exploring it in relation to a…

  10. Reclaiming hope: Affect, temporality, politics

    NARCIS (Netherlands)

    Taş, B.

    2016-01-01

    The critical task I take up in this research is to reconceptualize hope as an affective orientation in time, which requires remaining open to the risks that the unknowability of the future entails. I consider this opening a political contestation that is necessary to critique the current instrumenta

  11. A warm touch of affect?

    NARCIS (Netherlands)

    Willemse, Christian J.A.M.

    2015-01-01

    One of the research areas within affective Computer Mediated Communication currently under investigation is that of mediated social touch. A social touch is a complex composition of different physical parameters that can be simulated by haptic technologies. In this article we argue why we think it m

  12. Unconscious Affective Responses to Food

    Science.gov (United States)

    Sato, Wataru; Sawada, Reiko; Kubota, Yasutaka; Toichi, Motomi; Fushiki, Tohru

    2016-01-01

    Affective or hedonic responses to food are crucial for humans, both advantageously (e.g., enhancing survival) and disadvantageously (e.g., promoting overeating and lifestyle-related disease). Although previous psychological studies have reported evidence of unconscious cognitive and behavioral processing related to food, it remains unknown whether affective reactions to food can be triggered unconsciously and its relationship with daily eating behaviors. We investigated these issues by using the subliminal affective priming paradigm. Photographs of food or corresponding mosaic images were presented in the peripheral visual field for 33 ms. Target photos of faces with emotionally neutral expressions were then presented, and participants rated their preferences for the faces. Eating behaviors were also assessed using questionnaires. The food images, relative to the mosaics, increased participants’ preference for subsequent target faces. Furthermore, the difference in the preference induced by food versus mosaic images was positively correlated with the tendency to engage in external eating. These results suggest that unconscious affective reactions are elicited by the sight of food and that these responses contribute to daily eating behaviors related to overeating. PMID:27501443

  13. Food aroma affects bite size

    Directory of Open Access Journals (Sweden)

    de Wijk René A

    2012-03-01

    Full Text Available Abstract Background To evaluate the effect of food aroma on bite size, a semisolid vanilla custard dessert was delivered repeatedly into the mouth of test subjects using a pump while various concentrations of cream aroma were presented retronasally to the nose. Termination of the pump, which determined bite size, was controlled by the subject via a push button. Over 30 trials with 10 subjects, the custard was presented randomly either without an aroma, or with aromas presented below or near the detection threshold. Results Results for ten subjects (four females and six males, aged between 26 and 50 years, indicated that aroma intensity affected the size of the corresponding bite as well as that of subsequent bites. Higher aroma intensities resulted in significantly smaller sizes. Conclusions These results suggest that bite size control during eating is a highly dynamic process affected by the sensations experienced during the current and previous bites.

  14. Psychological factors affecting equine performance

    Directory of Open Access Journals (Sweden)

    McBride Sebastian D

    2012-09-01

    Full Text Available Abstract For optimal individual performance within any equestrian discipline horses must be in peak physical condition and have the correct psychological state. This review discusses the psychological factors that affect the performance of the horse and, in turn, identifies areas within the competition horse industry where current behavioral research and established behavioral modification techniques could be applied to further enhance the performance of animals. In particular, the role of affective processes underpinning temperament, mood and emotional reaction in determining discipline-specific performance is discussed. A comparison is then made between the training and the competition environment and the review completes with a discussion on how behavioral modification techniques and general husbandry can be used advantageously from a performance perspective.

  15. Salience-Affected Neural Networks

    CERN Document Server

    Remmelzwaal, Leendert A; Ellis, George F R

    2010-01-01

    We present a simple neural network model which combines a locally-connected feedforward structure, as is traditionally used to model inter-neuron connectivity, with a layer of undifferentiated connections which model the diffuse projections from the human limbic system to the cortex. This new layer makes it possible to model global effects such as salience, at the same time as the local network processes task-specific or local information. This simple combination network displays interactions between salience and regular processing which correspond to known effects in the developing brain, such as enhanced learning as a result of heightened affect. The cortex biases neuronal responses to affect both learning and memory, through the use of diffuse projections from the limbic system to the cortex. Standard ANNs do not model this non-local flow of information represented by the ascending systems, which are a significant feature of the structure of the brain, and although they do allow associational learning with...

  16. Environmental issues affecting CCT development

    Energy Technology Data Exchange (ETDEWEB)

    Reidy, M. [U.S. House of Representatives, Washington, DC (United States)

    1997-12-31

    While no final legislative schedule has been set for the new Congress, two issues with strong environmental ramifications which are likely to affect the coal industry seem to top the list of closely watched debates in Washington -- the Environmental Protection Agency`s proposed new ozone and particulate matter standards and utility restructuring. The paper discusses the background of the proposed standards, public comment, the Congressional review of regulations, other legislative options, and utility restructuring.

  17. Rodent empathy and affective neuroscience.

    Science.gov (United States)

    Panksepp, Jules B; Lahvis, Garet P

    2011-10-01

    In the past few years, several experimental studies have suggested that empathy occurs in the social lives of rodents. Thus, rodent behavioral models can now be developed to elucidate the mechanistic substrates of empathy at levels that have heretofore been unavailable. For example, the finding that mice from certain inbred strains express behavioral and physiological responses to conspecific distress, while others do not, underscores that the genetic underpinnings of empathy are specifiable and that they could be harnessed to develop new therapies for human psychosocial impairments. However, the advent of rodent models of empathy is met at the outset with a number of theoretical and semantic problems that are similar to those previously confronted by studies of empathy in humans. The distinct underlying components of empathy must be differentiated from one another and from lay usage of the term. The primary goal of this paper is to review a set of seminal studies that are directly relevant to developing a concept of empathy in rodents. We first consider some of the psychological phenomena that have been associated with empathy, and within this context, we consider the component processes, or endophenotypes of rodent empathy. We then review a series of recent experimental studies that demonstrate the capability of rodents to detect and respond to the affective state of their social partners. We focus primarily on experiments that examine how rodents share affective experiences of fear, but we also highlight how similar types of experimental paradigms can be utilized to evaluate the possibility that rodents share positive affective experiences. Taken together, these studies were inspired by Jaak Panksepp's theory that all mammals are capable of felt affective experiences.

  18. The Affections of My Life

    Institute of Scientific and Technical Information of China (English)

    Zhang; Yan; Shi; Xiao; jing

    2013-01-01

    <正>When I look back over the 90 years of my life, through all the tumultuous events, highs and lows, joys and sorrows, I see that one bright, shining emotion has always warmed my heart: affection. The pillar supporting me throughout has been family love: the care of my parents, the love of my wife and children, and the close feelings between myself and my

  19. Emotional processing affects movement speed.

    Science.gov (United States)

    Hälbig, Thomas D; Borod, Joan C; Frisina, Pasquale G; Tse, Winona; Voustianiouk, Andrei; Olanow, C Warren; Gracies, Jean-Michel

    2011-09-01

    Emotions can affect various aspects of human behavior. The impact of emotions on behavior is traditionally thought to occur at central, cognitive and motor preparation stages. Using EMG to measure the effects of emotion on movement, we found that emotional stimuli differing in valence and arousal elicited highly specific effects on peripheral movement time. This result has conceptual implications for the emotion-motion link and potentially practical implications for neurorehabilitation and professional environments where fast motor reactions are critical.

  20. Affective robot for elderly assistance.

    Science.gov (United States)

    Carelli, Laura; Gaggioli, Andrea; Pioggia, Giovanni; De Rossi, Federico; Riva, Giuseppe

    2009-01-01

    Recently, several robotic solutions for the elderly have been proposed. However, to date, the diffusion of these devices has been limited: available robots are too cumbersome, awkward, and expensive to become widely adopted. Another key issue which reduces the appeal of assistive robots is the lack of socio-emotional interaction: affective interchanges represent key requirements to create sustainable relationships between elderly and robots. In this paper, we propose a new approach to enhance the acceptability of robotic systems, based on the introduction of affective dimensions in human-robot interaction. This strategy is aimed at designing a new generation of relational and cognitive robots fusing information from embodied unobtrusive sensory interfaces. The final objective is to develop embodied interfaces, which are able to learn and adapt their affective responses to the user's behavior. User and robot will engage in natural interactions, involving verbal and non-verbal communication, improving empathic exchange of moods and feelings. Relevant independent living and quality of life related issues will be addressed: on-going monitoring of health parameters, assistance in everyday's activities, social support and cognitive/physical exercises. We expect that the proposed strategy will enhance the user's acceptance and adoption of the assistive robotic system.

  1. Musical affect regulation in infancy.

    Science.gov (United States)

    Trehub, Sandra E; Ghazban, Niusha; Corbeil, Mariève

    2015-03-01

    Adolescents and adults commonly use music for various forms of affect regulation, including relaxation, revitalization, distraction, and elicitation of pleasant memories. Mothers throughout the world also sing to their infants, with affect regulation as the principal goal. To date, the study of maternal singing has focused largely on its acoustic features and its consequences for infant attention. We describe recent laboratory research that explores the consequences of singing for infant affect regulation. Such work reveals that listening to recordings of play songs can maintain 6- to 9-month-old infants in a relatively contented or neutral state considerably longer than recordings of infant-directed or adult-directed speech. When 10-month-old infants fuss or cry and are highly aroused, mothers' multimodal singing is more effective than maternal speech at inducing recovery from such distress. Moreover, play songs are more effective than lullabies at reducing arousal in Western infants. We explore the implications of these findings along with possible practical applications.

  2. TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination

    Science.gov (United States)

    Mao, Zhiyong; Seluanov, Andrei; Jiang, Ying; Gorbunova, Vera

    2007-01-01

    TRF2 (telomeric repeat binding factor 2) is an essential component of the telomeric cap, where it forms and stabilizes the T-loop junctions. TRF2 forms the T-loops by stimulating strand invasion of the 3′ overhang into duplex DNA. TRF2 also has been shown to localize to nontelomeric DNA double-strand breaks, but its functional role in DNA repair has not been examined. Here, we present evidence that TRF2 is involved in homologous recombination (HR) repair of nontelomeric double-strand breaks. Depletion of TRF2 strongly inhibited HR and delayed the formation of Rad51 foci after γ-irradiation, whereas overexpression of TRF2 stimulated HR. Depletion of TRF2 had no effect on nonhomologous end-joining, and overexpression of TRF2 inhibited nonhomologous end-joining. We propose, based on our results and on the ability of TRF2 to mediate strand invasion, that TRF2 plays an essential role in HR by facilitating the formation of early recombination intermediates. PMID:17670947

  3. Deficiency in Homologous Recombination Renders Mammalian Cells More Sensitive to Proton Versus Photon Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Grosse, Nicole; Fontana, Andrea O. [Laboratory for Molecular Radiobiology, University Hospital Zurich, Zurich (Switzerland); Hug, Eugen B.; Lomax, Antony; Coray, Adolf [Center for Proton Therapy, Paul Scherrer Institute, Villigen (Switzerland); Augsburger, Marc [Laboratory for Molecular Radiobiology, University Hospital Zurich, Zurich (Switzerland); Paganetti, Harald [Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Sartori, Alessandro A. [Institute of Molecular Cancer Research, University of Zurich, Zurich (Switzerland); Pruschy, Martin, E-mail: martin.pruschy@usz.ch [Laboratory for Molecular Radiobiology, University Hospital Zurich, Zurich (Switzerland)

    2014-01-01

    Purpose: To investigate the impact of the 2 major DNA repair machineries on cellular survival in response to irradiation with the 2 types of ionizing radiation. Methods and Materials: The DNA repair and cell survival endpoints in wild-type, homologous recombination (HR)-deficient, and nonhomologous end-joining-deficient cells were analyzed after irradiation with clinically relevant, low-linear energy transfer (LET) protons and 200-keV photons. Results: All cell lines were more sensitive to proton irradiation compared with photon irradiation, despite no differences in the induction of DNA breaks. Interestingly, HR-deficient cells and wild-type cells with small interfering RNA-down-regulated Rad51 were markedly hypersensitive to proton irradiation, resulting in an increased relative biological effectiveness in comparison with the relative biological effectiveness determined in wild-type cells. In contrast, lack of nonhomologous end-joining did not result in hypersensitivity toward proton irradiation. Repair kinetics of DNA damage in wild-type cells were equal after both types of irradiation, although proton irradiation resulted in more lethal chromosomal aberrations. Finally, repair kinetics in HR-deficient cells were significantly delayed after proton irradiation, with elevated amounts of residual γH2AX foci after irradiation. Conclusion: Our data indicate a differential quality of DNA damage by proton versus photon irradiation, with a specific requirement for homologous recombination for DNA repair and enhanced cell survival. This has potential relevance for clinical stratification of patients carrying mutations in the DNA damage response pathways.

  4. DNA-dependent protein kinase regulates DNA end resection in concert with Mre11-Rad50-Nbs1 (MRN) and ataxia telangiectasia-mutated (ATM).

    Science.gov (United States)

    Zhou, Yi; Paull, Tanya T

    2013-12-27

    The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.

  5. UbcH7 regulates 53BP1 stability and DSB repair.

    Science.gov (United States)

    Han, Xiangzi; Zhang, Lei; Chung, Jinsil; Mayca Pozo, Franklin; Tran, Amanda; Seachrist, Darcie D; Jacobberger, James W; Keri, Ruth A; Gilmore, Hannah; Zhang, Youwei

    2014-12-09

    DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.

  6. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    Science.gov (United States)

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.

  7. Biological effects of exposure to intermediate neutron and repair mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Utsumi, Hiroshi; Sasaki, Masao [Kyoto Univ. (Japan); Onishi, Takeo; Onizuka, Masahiko

    2000-01-01

    An investigation was made on cytotoxic effects of neutron capture using chicken B-cell line mutants, DT40, KURO{sup -/-}, RAD54 {sup -/-} and KU70{sup -/-} / RAD54{sup -/-}. Suspensions of these cells were exposed to two times X-radiation at various doses and the cell surviving was evaluated. The sensitivity to radiation was highest in the double defective mutant, KU70{sup -/-} / RAD54{sup -/-} and followed by that of RAD54 {sup -/-}, a homologous recombination mutant, whereas KURO {sup -/-} cell, a non-homologous end-joining mutant showed a peculiar surviving curve composed of two phases and the cell was highly sensitive to a low-dose radiation. This indicates that there are two different DNA repair systems for double-strand breaks and the system for non-homologous end-joining repair can be involved in all phases of cell cycle, but the system for the homologous one is involved only in S-phase. Therefore, it was thought that variation of sensitivity to radiation exposure depending to the phase of cell cycle might explain the alternation of repair system depending to the phase progressing of cell cycle. It was thus likely that the recovery from radiation injury, which is still a black box might be explained with the double strand breaks of DNA. (M.N.)

  8. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    Science.gov (United States)

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  9. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    Energy Technology Data Exchange (ETDEWEB)

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  10. Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus.

    Science.gov (United States)

    Hayata, Koutarou; Asada, Seiya; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Sawayama, Shigeki

    2014-11-01

    The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

  11. Zinc finger nucleases and their application%锌指核酶技术的原理及应用

    Institute of Scientific and Technical Information of China (English)

    邓珊珊; 王颖芝; 马端

    2010-01-01

    锌指核酶(zinc finger nucleases,ZFNs)是将锌指蛋白的DNA识别域和非特异性核酸内切酶FokⅠ人工连接而构成的一种核酶.1对ZFNs能在DNA上产生双链断裂(double-strand breaks,DSB),诱导细胞发生同源重组(homology recombination,HR)或非同源末端连接(nonhomologous end joining,NHEJ).最近锌指核酶技术在基因功能的研究中受到重视.作者就ZFNs的作用原理、关键技术及其应用领域进行介绍.%Zinc finger nuclease (ZFN), which is a chimeric fusion structure between a Cys2-His2 zinc-finger protein (ZFP) and the cleavage domain of Fok Ⅰ endonuclease,can be used to introduce targeted double-stranded breaks (DSBs). ZFN-mediated cleavage leads to mutations when double-stranded breaks are repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ). In recent years, ZFNs are widely used in the fields of genetic research. In this review, the methodology and technical advantages of ZFNs were briefly discussed.

  12. Psychological Factors Affecting Infertile Women

    Directory of Open Access Journals (Sweden)

    Sati Unal

    2010-10-01

    Full Text Available AIM: This study was performed to determine the psychological factors affecting infertile women presenting at the infertility outpatients department. METHOD: The sample of this cross-sectional study consisted of 344 women who presented at the IVF center of a special branch hospital or a university hospital, March 2008 through September 2008, as determined by the non-random sampling method. All participating women gave their informed consent. The data were collected using the Data Form that consisted of questions on socio-demographic features and the Infertility Distress Scale (IDS. In the results, percentages were provided along with the Kruskal-Wallis H, Mann-Whitney U and Spearman correlation tests. RESULTS: The mean IDS was 39.01±9.6. There was a statistically significant linear relationship between the mean IDS score and age (r=0.106, p=0.048, marriage duration (r=0.232, p<0.001 and duration of desire to have a child (r=0.217, p<0.001. Women who were primary school graduates (X²=13.03, p=0.004, did not work (p=0.007, had no social security benefits (p=0.021 or from low socioeconomic status (X²=24.85, p<0.001 had significantly higher mean IDS scores. CONCLUSION: The results of this study show women become more adversely affected by infertility as their age, duration of marriage, and duration of desire to have a child increase. Women who are primary school graduates, do not work, have no social security benefits or have lower income are affected more negatively. We believe that taking these features into account when evaluating and planning supportive approaches for women presenting at the infertility treatment center and determining the psychological state of the women using the IDS will increase treatment success. [TAF Prev Med Bull 2010; 9(5.000: 481-486

  13. Does trade affect child health?

    Science.gov (United States)

    Levine, David I; Rothman, Dov

    2006-05-01

    Frankel and Romer [Frankel, J., Romer, D., 1999. Does trade cause growth? American Economic Review 89 (3), 379-399] documented positive effects of geographically determined trade openness on economic growth. At the same time, critics fear that openness can lead to a "race to the bottom" that increases pollution and reduces government resources for investments in health and education. We use Frankel and Romer's gravity model of trade to examine how openness to trade affects children. Overall, we find little harm from trade, and potential benefits largely through slightly faster GDP growth.

  14. affective variables of language learning

    Institute of Scientific and Technical Information of China (English)

    李文敬

    2011-01-01

    why people enjoy different degrees of success in second language learning,given similar opportunities.in the presence of overly negative emotions such as anxiety,fear,stress,anger or depression,our optimal learning potential maybe compromised.the affective domain refers to the emotional domain that has to do with the emotional behavior of human beings.it includes such factors as self-confidence,extroversion,anxiety,attitudes and motivation.three major factors are introduced here:self-confidence,anxiety and motivation.

  15. miR-155 Over-expression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-prone DSB Repair

    Science.gov (United States)

    Czochor, Jennifer R.; Sulkowski, Parker; Glazer, Peter M.

    2016-01-01

    miR-155 is an oncogenic microRNA (miR) that is often over-expressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors including RAD51, MLH1, and MSH6, and its over-expression results in an increased mutation frequency in vitro, although the mechanism has yet to be fully understood. Here, we demonstrate that over-expression of miR-155 drives an increased mutation frequency both in vitro and in vivo, promoting genomic instability by affecting multiple DNA repair pathways. miR-155 over-expression causes a decrease in homologous recombination, but yields a concurrent increase in the error-prone non-homologous end-joining (NHEJ) pathway. Despite repressing established targets MLH1 and MSH6, the identified mutation pattern upon miR-155 over-expression does not resemble that of a mismatch repair-deficient background. Further investigation revealed that all four subunits of polymerase delta, a high-fidelity DNA replication and repair polymerase, are down-regulated at the mRNA level in the context of miR-155 over-expression. FOXO3a, a transcription factor and known target of miR-155, has one or more putative binding site(s) in the promoter of all four polymerase delta subunits. Finally, suppression of FOXO3a by miR-155 or by siRNA knockdown is sufficient to repress the expression of the catalytic subunit of polymerase delta, POLD1, at the protein level, indicating that FOXO3a contributes to the regulation of polymerase delta levels. PMID:26850462

  16. Artemisinin–Second Career as Anticancer Drug?

    Directory of Open Access Journals (Sweden)

    Thomas Efferth

    2015-10-01

    Full Text Available Artemisinin represents a showcase example not only for the activity of medicinal herbs deriving from traditional chinese medicine, but for phytotherapy in general. Its isolation from Sweet Wormwood (qinhao, Artemisia annua L. represents the starting point for an unprecedent success story in the treatment of malaria worldwide. Beyond the therapeutic value against Plasmodium parasites, it turned out in recent years that the bioactivity of artemisinin is not restricted to malaria. We and others found that this sesquiterpenoid also exerts profound anticancer activity in vitro and in vivo. Artemisinin-type drugs exert multi-factorial cellular and molecular actions in cancer cells. Ferrous iron reacts with artemisinin, which leads to the formation of reactive oxygen species and ultimately to a plethora anticancer effects of artemisinins, e.g. expression of antioxidant response genes, cell cycle arrest (G1 as well as G2 phase arrests, DNA damage that is repaird by base excision repair, homogous recombination and non-homologous end-joining, as well as different modes of cell death (intrinsic and extrinsic apoptosis, autophagy, necrosis, necroptosis, oncosis, and ferroptosis. Furthermore, artemisinins inhibit neoangiogenesis in tumors. The signaling of major transcription factors (NF-κB, MYC/MAX, AP-1, CREBP, mTOR etc. and signaling pathways are affected by artemisinins (e.g. Wnt/β-catenin pathway, AMPK pathway, metastatic pathways, nitric oxide signaling, and others. Several case reports on the compassionate use of artemisinins as well as clinical Phase I/II pilot studies indicate the clinical activity of artemisinins in veterinary and human cancer patients. Larger scale of Phase II and III clinical studies are required now to further develop artemisinin-type compounds as novel anticancer drugs.

  17. DCLRE1C (ARTEMIS) mutations causing phenotypes ranging from atypical severe combined immunodeficiency to mere antibody deficiency

    Science.gov (United States)

    Volk, Timo; Pannicke, Ulrich; Reisli, Ismail; Bulashevska, Alla; Ritter, Julia; Björkman, Andrea; Schäffer, Alejandro A.; Fliegauf, Manfred; Sayar, Esra H.; Salzer, Ulrich; Fisch, Paul; Pfeifer, Dietmar; Di Virgilio, Michela; Cao, Hongzhi; Yang, Fang; Zimmermann, Karin; Keles, Sevgi; Caliskaner, Zafer; Güner, S¸ükrü; Schindler, Detlev; Hammarström, Lennart; Rizzi, Marta; Hummel, Michael; Pan-Hammarström, Qiang; Schwarz, Klaus; Grimbacher, Bodo

    2015-01-01

    Null mutations in genes involved in V(D)J recombination cause a block in B- and T-cell development, clinically presenting as severe combined immunodeficiency (SCID). Hypomorphic mutations in the non-homologous end-joining gene DCLRE1C (encoding ARTEMIS) have been described to cause atypical SCID, Omenn syndrome, Hyper IgM syndrome and inflammatory bowel disease—all with severely impaired T-cell immunity. By whole-exome sequencing, we investigated the molecular defect in a consanguineous family with three children clinically diagnosed with antibody deficiency. We identified perfectly segregating homozygous variants in DCLRE1C in three index patients with recurrent respiratory tract infections, very low B-cell numbers and serum IgA levels. In patients, decreased colony survival after irradiation, impaired proliferative response and reduced counts of naïve T cells were observed in addition to a restricted T-cell receptor repertoire, increased palindromic nucleotides in the complementarity determining regions 3 and long stretches of microhomology at switch junctions. Defective V(D)J recombination was complemented by wild-type ARTEMIS protein in vitro. Subsequently, homozygous or compound heterozygous DCLRE1C mutations were identified in nine patients from the same geographic region. We demonstrate that DCLRE1C mutations can cause a phenotype presenting as only antibody deficiency. This novel association broadens the clinical spectrum associated with ARTEMIS mutations. Clinicians should consider the possibility that an immunodeficiency with a clinically mild initial presentation could be a combined immunodeficiency, so as to provide appropriate care for affected patients. PMID:26476407

  18. Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway.

    Directory of Open Access Journals (Sweden)

    David Faucher

    2010-08-01

    Full Text Available Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3, both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells.

  19. Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Zhiming [Department of Neurosurgery, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan (China); Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Ping; Wang Hongyan; Zhang Xiangming [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Minli [Division of Life Sciences, Universities Space Research Association, Houston, Texas (United States); Cucinotta, Francis A. [National Aeronautics and Space Administration, Lyndon B. Johnson Space Center, Houston, Texas (United States); Wang Ya, E-mail: ywang94@emory.edu [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)

    2013-02-01

    Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials: Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.

  20. Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes.

    Science.gov (United States)

    Zhu, P; Liu, Q; Liu, S; Su, X; Feng, W; Lei, X; Liu, J; Cui, K; Huang, B; Shi, D

    2015-06-01

    In this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3-targeted mice were generated by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial bio-companies. The TALENs mutagenic non-homologous end-joining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK-293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN-mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN-mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3-independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3(-/+) ), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide-type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENs-based gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.

  1. Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway.

    Science.gov (United States)

    Faucher, David; Wellinger, Raymund J

    2010-08-26

    Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3), both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB) repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells.

  2. Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines.

    Science.gov (United States)

    Nishida, Yayoi; Mizutani, Naoki; Inoue, Minami; Omori, Yukari; Tamiya-Koizumi, Keiko; Takagi, Akira; Kojima, Tetsuhito; Suzuki, Motoshi; Nozawa, Yoshinori; Minami, Yosuke; Ohnishi, Kazunori; Naoe, Tomoki; Murate, Takashi

    2014-01-01

    Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

  3. Human longevity and variation in DNA damage response and repair: study of the contribution of sub-processes using competitive gene-set analysis.

    Science.gov (United States)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike; Dato, Serena; Mengel-From, Jonas; Stevnsner, Tinna; Bohr, Vilhelm A; Kruse, Torben A; Schreiber, Stefan; Nebel, Almut; Christensen, Kaare; Tan, Qihua; Christiansen, Lene

    2014-09-01

    DNA-damage response and repair are crucial to maintain genetic stability, and are consequently considered central to aging and longevity. Here, we investigate whether this pathway overall associates to longevity, and whether specific sub-processes are more strongly associated with longevity than others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10(-5)), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using the competitive gene-set analysis by Wang et al indicated that BER, HRR and RECQ associated stronger with longevity than the respective remaining genes of the pathway (P-values=0.004-0.048). For HRR and RECQ, only one gene contributed to the significance, whereas for BER several genes contributed. These associations did, however, generally not pass correction for multiple testing. Still, these findings indicate that, of the entire pathway, variation in BER might influence longevity the most. These modest sized P-values were not replicated in a German sample. This might, though, be due to differences in genotyping procedures and investigated SNPs, potentially inducing differences in the coverage of gene regions. Specifically, five genes were not covered at all in the German data. Therefore, investigations in additional study populations are needed before final conclusion can be drawn.

  4. Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

    Directory of Open Access Journals (Sweden)

    Hua Fung

    Full Text Available Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR or nonhomologous end-joining (NHEJ. For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

  5. DCLRE1C (ARTEMIS) mutations causing phenotypes ranging from atypical severe combined immunodeficiency to mere antibody deficiency.

    Science.gov (United States)

    Volk, Timo; Pannicke, Ulrich; Reisli, Ismail; Bulashevska, Alla; Ritter, Julia; Björkman, Andrea; Schäffer, Alejandro A; Fliegauf, Manfred; Sayar, Esra H; Salzer, Ulrich; Fisch, Paul; Pfeifer, Dietmar; Di Virgilio, Michela; Cao, Hongzhi; Yang, Fang; Zimmermann, Karin; Keles, Sevgi; Caliskaner, Zafer; Güner, S Ükrü; Schindler, Detlev; Hammarström, Lennart; Rizzi, Marta; Hummel, Michael; Pan-Hammarström, Qiang; Schwarz, Klaus; Grimbacher, Bodo

    2015-12-20

    Null mutations in genes involved in V(D)J recombination cause a block in B- and T-cell development, clinically presenting as severe combined immunodeficiency (SCID). Hypomorphic mutations in the non-homologous end-joining gene DCLRE1C (encoding ARTEMIS) have been described to cause atypical SCID, Omenn syndrome, Hyper IgM syndrome and inflammatory bowel disease-all with severely impaired T-cell immunity. By whole-exome sequencing, we investigated the molecular defect in a consanguineous family with three children clinically diagnosed with antibody deficiency. We identified perfectly segregating homozygous variants in DCLRE1C in three index patients with recurrent respiratory tract infections, very low B-cell numbers and serum IgA levels. In patients, decreased colony survival after irradiation, impaired proliferative response and reduced counts of naïve T cells were observed in addition to a restricted T-cell receptor repertoire, increased palindromic nucleotides in the complementarity determining regions 3 and long stretches of microhomology at switch junctions. Defective V(D)J recombination was complemented by wild-type ARTEMIS protein in vitro. Subsequently, homozygous or compound heterozygous DCLRE1C mutations were identified in nine patients from the same geographic region. We demonstrate that DCLRE1C mutations can cause a phenotype presenting as only antibody deficiency. This novel association broadens the clinical spectrum associated with ARTEMIS mutations. Clinicians should consider the possibility that an immunodeficiency with a clinically mild initial presentation could be a combined immunodeficiency, so as to provide appropriate care for affected patients.

  6. Linear Energy Transfer-Dependent Change in Rice Gene Expression Profile after Heavy-Ion Beam Irradiation.

    Directory of Open Access Journals (Sweden)

    Kotaro Ishii

    Full Text Available A heavy-ion beam has been recognized as an effective mutagen for plant breeding and applied to the many kinds of crops including rice. In contrast with X-ray or γ-ray, the heavy-ion beam is characterized by a high linear energy transfer (LET. LET is an important factor affecting several aspects of the irradiation effect, e.g. cell survival and mutation frequency, making the heavy-ion beam an effective mutagen. To study the mechanisms behind LET-dependent effects, expression profiling was performed after heavy-ion beam irradiation of imbibed rice seeds. Array-based experiments at three time points (0.5, 1, 2 h after the irradiation revealed that the number of up- or down-regulated genes was highest 2 h after irradiation. Array-based experiments with four different LETs at 2 h after irradiation identified LET-independent regulated genes that were up/down-regulated regardless of the value of LET; LET-dependent regulated genes, whose expression level increased with the rise of LET value, were also identified. Gene ontology (GO analysis of LET-independent up-regulated genes showed that some GO terms were commonly enriched, both 2 hours and 3 weeks after irradiation. GO terms enriched in LET-dependent regulated genes implied that some factor regulates genes that have kinase activity or DNA-binding activity in cooperation with the ATM gene. Of the LET-dependent up-regulated genes, OsPARP3 and OsPCNA were identified, which are involved in DNA repair pathways. This indicates that the Ku-independent alternative non-homologous end-joining pathway may contribute to repairing complex DNA legions induced by high-LET irradiation. These findings may clarify various LET-dependent responses in rice.

  7. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    Science.gov (United States)

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  8. Mood Swings: An Affective Interactive Art System

    Science.gov (United States)

    Bialoskorski, Leticia S. S.; Westerink, Joyce H. D. M.; van den Broek, Egon L.

    The progress in the field of affective computing enables the realization of affective consumer products, affective games, and affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on the integration of a framework for affective movements and a color model. This enables Mood Swings to recognize affective movement characteristics as expressed by a person and display a color that matches the expressed emotion. With that, a unique interactive system is introduced, which can be considered as art, a game, or a combination of both.

  9. Pseudobulbar affect: prevalence and management

    Directory of Open Access Journals (Sweden)

    Ahmed A

    2013-11-01

    Full Text Available Aiesha Ahmed, Zachary SimmonsDepartment of Neurology, Penn State Hershey Medical Center, Hershey, PA, USAAbstract: Pseudobulbar affect (PBA may occur in association with a variety of neurological diseases, and so may be encountered in the setting of amyotrophic lateral sclerosis, extrapyramidal and cerebellar disorders, multiple sclerosis, traumatic brain injury, Alzheimer's disease, stroke, and brain tumors. The psychological consequences and the impact on social interactions may be substantial. Although it is most commonly misidentified as a mood disorder, particularly depression or a bipolar disorder, there are characteristic features that can be recognized clinically or assessed by validated scales, resulting in accurate identification of PBA, and thus permitting proper management and treatment. Mechanistically, PBA is a disinhibition syndrome in which pathways involving serotonin and glutamate are disrupted. This knowledge has permitted effective treatment for many years with antidepressants, particularly tricyclic antidepressants and selective serotonin reuptake inhibitors. A recent therapeutic breakthrough occurred with the approval by the Food and Drug Administration of a dextromethorphan/quinidine combination as being safe and effective for treatment of PBA. Side effect profiles and contraindications differ for the various treatment options, and the clinician must be familiar with these when choosing the best therapy for an individual, particularly elderly patients and those with multiple comorbidities and concomitant medications.Keywords: pseudobulbar affect, emotional lability, depression, amyotrophic lateral sclerosis, multiple sclerosis

  10. Rheumatoid arthritis affecting temporomandibular joint.

    Science.gov (United States)

    Sodhi, Amandeep; Naik, Shobha; Pai, Anuradha; Anuradha, Ardra

    2015-01-01

    Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune inflammatory disorder that is characterized by joint inflammation, erosive properties and symmetric multiple joint involvement. Temporomandibular joint (TMJ) is very rare to be affected in the early phase of the disease, thus posing diagnostic challenges for the dentist. Conventional radiographs fail to show the early lesions due to its limitations. More recently cone-beam computed tomography (CBCT) has been found to diagnose the early degenerative changes of TMJ and hence aid in the diagnosis of the lesions more accurately. Our case highlights the involvement of TMJ in RA and the role of advanced imaging (CBCT) in diagnosing the bony changes in the early phase of the disease.

  11. Bipolar Affective Disorder and Migraine

    Directory of Open Access Journals (Sweden)

    Birk Engmann

    2012-01-01

    Full Text Available This paper consists of a case history and an overview of the relationship, aetiology, and treatment of comorbid bipolar disorder migraine patients. A MEDLINE literature search was used. Terms for the search were bipolar disorder bipolar depression, mania, migraine, mood stabilizer. Bipolar disorder and migraine cooccur at a relatively high rate. Bipolar II patients seem to have a higher risk of comorbid migraine than bipolar I patients have. The literature on the common roots of migraine and bipolar disorder, including both genetic and neuropathological approaches, is broadly discussed. Moreover, bipolar disorder and migraine are often combined with a variety of other affective disorders, and, furthermore, behavioural factors also play a role in the origin and course of the diseases. Approach to treatment options is also difficult. Several papers point out possible remedies, for example, valproate, topiramate, which acts on both diseases, but no first-choice treatments have been agreed upon yet.

  12. Global Media, Biopolitics and Affect

    DEFF Research Database (Denmark)

    Knudsen, Britta Timm; Stage, Carsten

    Global Media, Biopolitics and Affect shows how mediations of bodily vulnerability have become a strong political force in contemporary societies. In discussions and struggles concerning war involvement, healthcare issues, charity, democracy movements, contested national pasts, and climate change...... particular political events or issues by means of media. To investigate how, when and why that happens, and to evaluate the long-term social impacts of mediating bodily vulnerability, the book offers a theoretical framework for understanding the role of bodily vulnerability in contemporary digital media...... culture. Likewise, it presents a range of close empirical case studies in the areas of illness blogging, global protests after the killing of Neda Agda Soltan in Iran, charity communication, green media activism, online war commemoration and digital witnessing related to conflicts in Sarajevo and Ukraine....

  13. Rheumatoid arthritis affecting temporomandibular joint

    Directory of Open Access Journals (Sweden)

    Amandeep Sodhi

    2015-01-01

    Full Text Available Rheumatoid arthritis (RA is a chronic, systemic, autoimmune inflammatory disorder that is characterized by joint inflammation, erosive properties and symmetric multiple joint involvement. Temporomandibular joint (TMJ is very rare to be affected in the early phase of the disease, thus posing diagnostic challenges for the dentist. Conventional radiographs fail to show the early lesions due to its limitations. More recently cone-beam computed tomography (CBCT has been found to diagnose the early degenerative changes of TMJ and hence aid in the diagnosis of the lesions more accurately. Our case highlights the involvement of TMJ in RA and the role of advanced imaging (CBCT in diagnosing the bony changes in the early phase of the disease.

  14. Affective and Cognitive Aspects of Bilingualism.

    Science.gov (United States)

    Herrell, Ileana Collado; Herrell, James M.

    1980-01-01

    A study showing that lexically equivalent words in two languages have different affective meanings, that affective intensity of dominant language words is greater than for second language equivalents, and that the difference is greatest for words with high affective meaning demonstrates that affective meaning is an important component of…

  15. The affective shift model of work engagement.

    Science.gov (United States)

    Bledow, Ronald; Schmitt, Antje; Frese, Michael; Kühnel, Jana

    2011-11-01

    On the basis of self-regulation theories, the authors develop an affective shift model of work engagement according to which work engagement emerges from the dynamic interplay of positive and negative affect. The affective shift model posits that negative affect is positively related to work engagement if negative affect is followed by positive affect. The authors applied experience sampling methodology to test the model. Data on affective events, mood, and work engagement was collected twice a day over 9 working days among 55 software developers. In support of the affective shift model, negative mood and negative events experienced in the morning of a working day were positively related to work engagement in the afternoon if positive mood in the time interval between morning and afternoon was high. Individual differences in positive affectivity moderated within-person relationships. The authors discuss how work engagement can be fostered through affect regulation.

  16. The Affective Regulation of Cognitive Priming

    OpenAIRE

    Storbeck, Justin; Clore, Gerald L.

    2008-01-01

    Semantic and affective priming are classic effects observed in cognitive and social psychology, respectively. We discovered that affect regulates such priming effects. In Experiment 1, positive and negative moods were induced prior to one of three priming tasks; evaluation, categorization, or lexical decision. As predicted, positive affect led to both affective priming (evaluation task) and semantic priming (category and lexical decision tasks). However, negative affect inhibited such effects...

  17. Twitter, Journalism and Affective Labour

    Directory of Open Access Journals (Sweden)

    Eugenia Siapera

    2015-03-01

    Full Text Available The rise of the network aspects of journalism in the context of social mediasuch as Twitter, and the increased importance accorded to community building and maintenance as well as to reciprocity, point to the need to take into account the affective part of journalistic labour. This refers to these aspects of journalistic work that are linked to the creation of networks and communities, to interactions with readers and the forming of bonds between journalists and their readers. An analysis of the affective labour of journalists on Twitter, we argue, is necessary in order to understand the potential and ambiguities of this part of their labour. Based on a set of in-depth interviews with Twitter journalists, this article found three main repertoires of affective labour: the organic relations repertoire, which points to the increasing importance of authenticity as a means of establishing credibility on Twitter; the temporal repertoire; and the repertoire of responsibility. The importance of the affective labour of journalism is found in its biopolitical productivity. The development of an organic relationship with followers, the emergence of stronger bonds between core groups that then become communities, the extension of care and help to the network, are all evidence of the importance of this biopolitical productivity and point to the construction of a new and potentially more radical sociopolitical role for journalism. However, this potential is ambiguous insofar as these elements contain unresolved tensions and ambiguities. These include the trade in selves and the associated commodification; the re-formulation of time, especially its diachronic dimension, as accumulation of social capital; the role of reciprocity and responsibility in reproducing inequalities; and care as care for only those deemed deserving. These ambiguities severely undermine and limit the potentials of affective labour, pointing to the need to develop a purposeful political

  18. Ash in fire affected ecosystems

    Science.gov (United States)

    Pereira, Paulo; Jordan, Antonio; Cerda, Artemi; Martin, Deborah

    2015-04-01

    Ash in fire affected ecosystems Ash lefts an important footprint in the ecosystems and has a key role in the immediate period after the fire (Bodi et al., 2014; Pereira et al., 2015). It is an important source of nutrients for plant recover (Pereira et al., 2014a), protects soil from erosion and controls soil hydrological process as runoff, infiltration and water repellency (Cerda and Doerr, 2008; Bodi et al., 2012, Pereira et al., 2014b). Despite the recognition of ash impact and contribution to ecosystems recuperation, it is assumed that we still have little knowledge about the implications of ash in fire affected areas. Regarding this situation we wanted to improve our knowledge in this field and understand the state of the research about fire ash around world. The special issue about "The role of ash in fire affected ecosystems" currently in publication in CATENA born from the necessity of joint efforts, identify research gaps, and discuss future cooperation in this interdisciplinary field. This is the first special issue about fire ash in the international literature. In total it will be published 10 papers focused in different aspects of the impacts of ash in fire affected ecosystems from several parts of the world: • Fire reconstruction using charcoal particles (Burjachs and Espositio, in press) • Ash slurries impact on rheological properties of Runoff (Burns and Gabet, in press) • Methods to analyse ash conductivity and sorbtivity in the laboratory and in the field (Balfour et al., in press) • Termogravimetric and hydrological properties of ash (Dlapa et al. in press) • Effects of ash cover in water infiltration (Leon et al., in press) • Impact of ash in volcanic soils (Dorta Almenar et al., in press; Escuday et al., in press) • Ash PAH and Chemical extracts (Silva et al., in press) • Microbiology (Barreiro et al., in press; Lombao et al., in press) We believe that this special issue will contribute importantly to the better understanding of

  19. Bilingualism affects audiovisual phoneme identification.

    Science.gov (United States)

    Burfin, Sabine; Pascalis, Olivier; Ruiz Tada, Elisa; Costa, Albert; Savariaux, Christophe; Kandel, Sonia

    2014-01-01

    We all go through a process of perceptual narrowing for phoneme identification. As we become experts in the languages we hear in our environment we lose the ability to identify phonemes that do not exist in our native phonological inventory. This research examined how linguistic experience-i.e., the exposure to a double phonological code during childhood-affects the visual processes involved in non-native phoneme identification in audiovisual speech perception. We conducted a phoneme identification experiment with bilingual and monolingual adult participants. It was an ABX task involving a Bengali dental-retroflex contrast that does not exist in any of the participants' languages. The phonemes were presented in audiovisual (AV) and audio-only (A) conditions. The results revealed that in the audio-only condition monolinguals and bilinguals had difficulties in discriminating the retroflex non-native phoneme. They were phonologically "deaf" and assimilated it to the dental phoneme that exists in their native languages. In the audiovisual presentation instead, both groups could overcome the phonological deafness for the retroflex non-native phoneme and identify both Bengali phonemes. However, monolinguals were more accurate and responded quicker than bilinguals. This suggests that bilinguals do not use the same processes as monolinguals to decode visual speech.

  20. Affective Priming Caused by Lying

    Directory of Open Access Journals (Sweden)

    Megumi Sato

    2011-10-01

    Full Text Available Typically, arousal increases when telling a lie, as indicated in psychophysiological studies about lie detection. But the emotional valence induced by lying is unknown, though intuition indicates that it may be negative. Indeed, the Electrodermal Activity (EDA, used in such studies, only shows arousal changes during an emotional response. In this study, we examined the emotional valence induced by lying using two tasks. First, in the deceptive task, participants answered “no” to every question regarding the nature of displayed playing cards. Therefore, they told a lie about specific cards. During the task, their EDA was recorded. Secondly, in the figure estimation task, they assessed pictures by “like” or “dislike” after looking at playing cards visibly or subliminally as prime stimuli. We expected them to tend to estimate figures by “dislike” when cards relevant to deception were previously shown. This would mean that an affective priming effect due to telling a lie happened. Actually, this effect was found only when prime stimuli were displayed visibly. This result suggests that lying per se induces negative emotions even without motivation or punishment due to lying. Furthermore, we found that such effect was more blatant in participants whose EDA changes were salient while lying.

  1. Factors affecting forage stand establishment

    Directory of Open Access Journals (Sweden)

    Sulc R.M.

    1998-01-01

    Full Text Available Significant advances have been made in our knowledge of forage seed physiology, technology, and stand establishment practices; however, stand establishment continues to be one of the most common production problems affecting forage crops in the USA. There is a need for research on stand establishment of forage crops under abiotic and biotic stress. Although the forage seed industry produces and markets seed of high quality, new methods of assessing seed vigor are needed and their use should be expanded in the industry to enable matching seed lot performance to specific environmental conditions where performance can be maximized. Seed treatment and seed coating are used in the forage seed industry, and studies have shown they are of benefit in some environments. There is an increase in no-tillage seeding of forage crops, but improvements in the no-tillage planting equipment are needed to make them better suited to small seeds. Other recent developments in seeding techniques include broadcasting seed with dry granular and fluid fertilizers, which improves the efficiency of the seeding operation.

  2. Southwest ballot measures affecting healthcare

    Directory of Open Access Journals (Sweden)

    Robbins RA

    2016-11-01

    Full Text Available No abstract available. Article truncated after 150 words. Modern Healthcare (1 has published an article summarizing ballot measures affecting healthcare. Those from the Southwest are listed below: States: Arizona: 1. Recreational marijuana. Proposition 205: Legalizes recreational marijuana use for people 21 and older. Opponents of the measure include the Arizona Health and Hospital Association and Insys Therapeutics, a company that makes a cannabis-based pain medication. California : 1. Medi-Cal hospital fee program. Proposition 52: Requires the legislature to get voter approval to use fee revenue for purposes other than generating federal matching funds and funding enhanced Medicaid payments and grants for hospitals. The initiative, which was written by the California Hospital Association and is supported by most state lawmakers, would also make the program permanent, requiring a supermajority in the legislature to end it. 2. Tobacco tax. Proposition 56: Increases the state's cigarette tax by $2 a pack and impose an "equivalent increase on other tobacco products and ...

  3. Factors Affecting Radiologist's PACS Usage.

    Science.gov (United States)

    Forsberg, Daniel; Rosipko, Beverly; Sunshine, Jeffrey L

    2016-12-01

    The purpose of this study was to determine if any of the factors radiologist, examination category, time of week, and week effect PACS usage, with PACS usage defined as the sequential order of computer commands issued by a radiologist in a PACS during interpretation and dictation. We initially hypothesized that only radiologist and examination category would have significant effects on PACS usage. Command logs covering 8 weeks of PACS usage were analyzed. For each command trace (describing performed activities of an attending radiologist interpreting a single examination), the PACS usage variables number of commands, number of command classes, bigram repetitiveness, and time to read were extracted. Generalized linear models were used to determine the significance of the factors on the PACS usage variables. The statistical results confirmed the initial hypothesis that radiologist and examination category affect PACS usage and that the factors week and time of week to a large extent have no significant effect. As such, this work provides direction for continued efforts to analyze system data to better understand PACS utilization, which in turn can provide input to enable optimal utilization and configuration of corresponding systems. These continued efforts were, in this work, exemplified by a more detailed analysis using PACS usage profiles, which revealed insights directly applicable to improve PACS utilization through modified system configuration.

  4. Factors affecting tactile spatial acuity.

    Science.gov (United States)

    Craig, J C; Kisner, J M

    1998-01-01

    Tactile spatial acuity on the fingerpad was measured using a grating orientation task. In this task, subjects are required to identify the orientation of square-wave gratings placed on the skin. Previous studies have shown that performance varies as a function of the width of the grooves in the gratings. In the present study, both groove width and the overall size and configuration of the contactors were varied. Sensitivity improved with wider grooves and with larger contactors. Additional measurements showed that the improved sensitivity is not the result of the increase in total area contacted, but rather is due to two other factors associated with larger contactors. One is the greater linear extent of the larger contactors. The other appears to be due to the reduction in the interference produced by the outer edge of the contactor. Specifically, as the contactor increases in size, the distance between the outer edge and the center portion of the grooves also increases. It was also shown that subjects are more sensitive to a single, continuous groove as compared with two grooves of the same total length but spatially discontinuous. Similarly, subjects are more sensitive to a contactor with a continuous groove than to a contactor in which just the end points of the groove are presented. The results are generally consistent with the results of peripheral, neurophysiological recordings. The results are discussed in terms of the way in which both spatial and intensive factors may affect sensitivity to grating orientation.

  5. Quantum Tunneling Affects Engine Performance.

    Science.gov (United States)

    Som, Sibendu; Liu, Wei; Zhou, Dingyu D Y; Magnotti, Gina M; Sivaramakrishnan, Raghu; Longman, Douglas E; Skodje, Rex T; Davis, Michael J

    2013-06-20

    We study the role of individual reaction rates on engine performance, with an emphasis on the contribution of quantum tunneling. It is demonstrated that the effect of quantum tunneling corrections for the reaction HO2 + HO2 = H2O2 + O2 can have a noticeable impact on the performance of a high-fidelity model of a compression-ignition (e.g., diesel) engine, and that an accurate prediction of ignition delay time for the engine model requires an accurate estimation of the tunneling correction for this reaction. The three-dimensional model includes detailed descriptions of the chemistry of a surrogate for a biodiesel fuel, as well as all the features of the engine, such as the liquid fuel spray and turbulence. This study is part of a larger investigation of how the features of the dynamics and potential energy surfaces of key reactions, as well as their reaction rate uncertainties, affect engine performance, and results in these directions are also presented here.

  6. How feeling betrayed affects cooperation.

    Directory of Open Access Journals (Sweden)

    Pouria Ramazi

    Full Text Available For a population of interacting self-interested agents, we study how the average cooperation level is affected by some individuals' feelings of being betrayed and guilt. We quantify these feelings as adjusted payoffs in asymmetric games, where for different emotions, the payoff matrix takes the structure of that of either a prisoner's dilemma or a snowdrift game. Then we analyze the evolution of cooperation in a well-mixed population of agents, each of whom is associated with such a payoff matrix. At each time-step, an agent is randomly chosen from the population to update her strategy based on the myopic best-response update rule. According to the simulations, decreasing the feeling of being betrayed in a portion of agents does not necessarily increase the level of cooperation in the population. However, this resistance of the population against low-betrayal-level agents is effective only up to some extend that is explicitly determined by the payoff matrices and the number of agents associated with these matrices. Two other models are also considered where the betrayal factor of an agent fluctuates as a function of the number of cooperators and defectors that she encounters. Unstable behaviors are observed for the level of cooperation in these cases; however, we show that one can tune the parameters in the function to make the whole population become cooperative or defective.

  7. Bilingualism affects audiovisual phoneme identification

    Directory of Open Access Journals (Sweden)

    Sabine eBurfin

    2014-10-01

    Full Text Available We all go through a process of perceptual narrowing for phoneme identification. As we become experts in the languages we hear in our environment we lose the ability to identify phonemes that do not exist in our native phonological inventory. This research examined how linguistic experience –i.e., the exposure to a double phonological code during childhood– affects the visual processes involved in non-native phoneme identification in audiovisual speech perception. We conducted a phoneme identification experiment with bilingual and monolingual adult participants. It was an ABX task involving a Bengali dental-retroflex contrast that does not exist in any of the participants’ languages. The phonemes were presented in audiovisual (AV and audio-only (A conditions. The results revealed that in the audio-only condition monolinguals and bilinguals had difficulties in discriminating the retroflex non-native phoneme. They were phonologically deaf and assimilated it to the dental phoneme that exists in their native languages. In the audiovisual presentation instead, both groups could overcome the phonological deafness for the retroflex non-native phoneme and identify both Bengali phonemes. However, monolinguals were more accurate and responded quicker than bilinguals. This suggests that bilinguals do not use the same processes as monolinguals to decode visual speech.

  8. Factors Affecting Aerosol Radiative Forcing

    Science.gov (United States)

    Wang, Jingxu; Lin, Jintai; Ni, Ruijing

    2016-04-01

    Rapid industrial and economic growth has meant a large amount of aerosols in the atmosphere with strong radiative forcing (RF) upon the climate system. Over parts of the globe, the negative forcing of aerosols has overcompensated for the positive forcing of greenhouse gases. Aerosol RF is determined by emissions and various chemical-transport-radiative processes in the atmosphere, a multi-factor problem whose individual contributors have not been well quantified. In this study, we analyze the major factors affecting RF of secondary inorganic aerosols (SIOAs, including sulfate, nitrate and ammonium), primary organic aerosol (POA), and black carbon (BC). We analyze the RF of aerosols produced by 11 major regions across the globe, including but not limited to East Asia, Southeast Asia, South Asia, North America, and Western Europe. Factors analyzed include population size, per capita gross domestic production (GDP), emission intensity (i.e., emissions per unit GDP), chemical efficiency (i.e., mass per unit emissions) and radiative efficiency (i.e., RF per unit mass). We find that among the 11 regions, East Asia produces the largest emissions and aerosol RF, due to relatively high emission intensity and a tremendous population size. South Asia produce the second largest RF of SIOA and BC and the highest RF of POA, in part due to its highest chemical efficiency among all regions. Although Southeast Asia also has large emissions, its aerosol RF is alleviated by its lowest chemical efficiency. The chemical efficiency and radiative efficiency of BC produced by the Middle East-North Africa are the highest across the regions, whereas its RF is lowered by a small per capita GDP. Both North America and Western Europe have low emission intensity, compensating for the effects on RF of large population sizes and per capita GDP. There has been a momentum to transfer industries to Southeast Asia and South Asia, and such transition is expected to continue in the coming years. The

  9. Dispositional affectivity and work outcomes of expatriates

    DEFF Research Database (Denmark)

    Selmer, Jan; Lauring, Jakob

    2013-01-01

    to examine the relationship between dispositional affectivity and their work outcomes. Results showed consistent positive associations between positive affectivity and all the studied work outcomes and the opposite relationships for negative affectivity. Implications and suggestions for future research......How the two components of dispositional affectivity, positive affectivity, representing the predisposition to respond positively to environmental stimuli, and negative affectivity, depicting the opposite reaction, influence work has been the focus of much research. Although dispositional...... affectivity appears to be a promising construct to explain and predict many attitudinal and behavioral outcomes in the workplace, few studies have empirically investigated dispositional affectivity and the work of expatriates. Hence, data from a net-based survey including 350 expatriates in Denmark were used...

  10. Common Problems That Can Affect Your Voice

    Science.gov (United States)

    ... near you Common Problems That Can Affect Your Voice Common Problems That Can Affect Your Voice Patient ... that traditionally accompany gastro esophageal reflux disease (GERD). Voice Misuse and Overuse Speaking is a physical task ...

  11. Light Therapy Boxes for Seasonal Affective Disorder

    Science.gov (United States)

    Diseases and Conditions Seasonal affective disorder (SAD) Light therapy boxes can offer an effective treatment for seasonal affective disorder. Features such as light intensity, safety, cost and style are important considerations. ...

  12. On English learning Disability and Affect

    Institute of Scientific and Technical Information of China (English)

    LIU Fang

    2007-01-01

    This paper introduces the definitions and causes of LD,pointing out that English LD is definitely concerned with the factor of affect by exploring the three theories like Affective Filter Hypothesis,Humanistic Approach to Education and Attribution Theory.

  13. Can the Weather Affect My Child's Asthma?

    Science.gov (United States)

    ... Year-Old Can the Weather Affect My Child's Asthma? KidsHealth > For Parents > Can the Weather Affect My ... empeorar el asma de mi hijo? Weather and Asthma The effect of weather on asthma symptoms isn' ...

  14. Antecedents and Consequences of Affective Commitment

    NARCIS (Netherlands)

    Bloemer, J.M.M.; Odekerken-Schröder, G.J.

    2003-01-01

    The primary objective of this study is to assess the impact of three psychological antecedents (position involvement, volitional choice and informational complexity) on affective commitment in a financial service setting. Furthermore, this study addresses the consequences of affective commitment on

  15. Affective Scaffolds, Expressive Arts, and Cognition

    OpenAIRE

    Maiese, Michelle

    2016-01-01

    Some theorists have argued that elements of the surrounding world play a crucial role in sustaining and amplifying both cognition and emotion. Such insights raise an interesting question about the relationship between cognitive and affective scaffolding: in addition to enabling the realization of specific affective states, can an affective niche also enable the realization of certain cognitive capacities? In order to gain a better understanding of this relationship between affective niches an...

  16. 40 CFR 62.14102 - Affected facilities.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Affected facilities. 62.14102 Section... (CONTINUED) APPROVAL AND PROMULGATION OF STATE PLANS FOR DESIGNATED FACILITIES AND POLLUTANTS Federal Plan... Affected facilities. (a) The affected facility to which this subpart applies is each municipal...

  17. An Affect Control Theory of Technology

    Science.gov (United States)

    Shank, Daniel B.

    2010-01-01

    Affect control theory is a theory of interaction that takes into account cultural meanings. Affect control research has previously considered interaction with technology, but there remains a lack of theorizing about inclusion of technology within the theory. This paper lays a foundation for an affect control theory of technology by addressing key…

  18. The effects of diffuse and distinct affect

    NARCIS (Netherlands)

    Stapel, D.A.; Koomen, W; Ruijs, K.I

    2002-01-01

    In a series of Suboptimal priming studies, it was shown that both affective and nonaffective reactions to a Stimulus may occur without awareness. Moreover. it was demonstrated that affective information is detected earlier than nonaffective information. Therefore, early reactions to an affect-laden

  19. Affective Priming with Associatively Acquired Valence

    Science.gov (United States)

    Aguado, Luis; Pierna, Manuel; Saugar, Cristina

    2005-01-01

    Three experiments explored the effect of affectively congruent or incongruent primes on evaluation responses to positive or negative valenced targets (the "affective priming" effect). Experiment 1 replicated the basic affective priming effect with Spanish nouns: reaction time for evaluative responses (pleasant/unpleasant) were slower on…

  20. Affect, Behavioural Schemas and the Proving Process

    Science.gov (United States)

    Selden, Annie; McKee, Kerry; Selden, John

    2010-01-01

    In this largely theoretical article, we discuss the relation between a kind of affect, behavioural schemas and aspects of the proving process. We begin with affect as described in the mathematics education literature, but soon narrow our focus to a particular kind of affect--nonemotional cognitive feelings. We then mention the position of feelings…

  1. Dynamic artificial neural networks with affective systems.

    Science.gov (United States)

    Schuman, Catherine D; Birdwell, J Douglas

    2013-01-01

    Artificial neural networks (ANNs) are processors that are trained to perform particular tasks. We couple a computational ANN with a simulated affective system in order to explore the interaction between the two. In particular, we design a simple affective system that adjusts the threshold values in the neurons of our ANN. The aim of this paper is to demonstrate that this simple affective system can control the firing rate of the ensemble of neurons in the ANN, as well as to explore the coupling between the affective system and the processes of long term potentiation (LTP) and long term depression (LTD), and the effect of the parameters of the affective system on its performance. We apply our networks with affective systems to a simple pole balancing example and briefly discuss the effect of affective systems on network performance.

  2. College English Listening Teaching Based on Affect

    Institute of Scientific and Technical Information of China (English)

    张冬梅

    2015-01-01

    With the continuous development of language teaching theories,the affective factors in language learning has got wide-spread attention.Research is carried out on the influence of learners'affective factors on the college English listening teaching,aiming to break the learning barricades caused by the affect.The theoretical background of affect and the current study situation abroad and domestically are introduced firstly,and together with the fact that learners'affect has widely been neglected in the teaching process.An interview and a questionnaire are used to test learners'affect in English listening class.In the end,how to solve the problems in college English listening teaching from the aspect of affect is discussed.

  3. Audio-visual affective expression recognition

    Science.gov (United States)

    Huang, Thomas S.; Zeng, Zhihong

    2007-11-01

    Automatic affective expression recognition has attracted more and more attention of researchers from different disciplines, which will significantly contribute to a new paradigm for human computer interaction (affect-sensitive interfaces, socially intelligent environments) and advance the research in the affect-related fields including psychology, psychiatry, and education. Multimodal information integration is a process that enables human to assess affective states robustly and flexibly. In order to understand the richness and subtleness of human emotion behavior, the computer should be able to integrate information from multiple sensors. We introduce in this paper our efforts toward machine understanding of audio-visual affective behavior, based on both deliberate and spontaneous displays. Some promising methods are presented to integrate information from both audio and visual modalities. Our experiments show the advantage of audio-visual fusion in affective expression recognition over audio-only or visual-only approaches.

  4. Factors Affecting University Library Website Design

    OpenAIRE

    Kim, Yongi-Mi; University of Oklahoma

    2011-01-01

    Existing studies have extensively explored factors that affect users’ intentions to use university library website resources (ULWR); yet little attention has been given to factors affecting university library website design. This paper investigates factors that affect university library website design and assesses the success of the university library website from both designers’ and users’ perspectives. The findings show that when planning a website, university web designers consider univers...

  5. Chromosome analysis of arsenic affected cattle

    Directory of Open Access Journals (Sweden)

    S. Shekhar

    2014-10-01

    Full Text Available Aim: The aim was to study the chromosome analysis of arsenic affected cattle. Materials and Methods: 27 female cattle (21 arsenic affected and 6 normal were selected for cytogenetical study. The blood samples were collected, incubated, and cultured using appropriate media and specific methods. The samples were analyzed for chromosome number and morphology, relative length of the chromosome, arm ratio, and centromere index of X chromosome and chromosomal abnormalities in arsenic affected cattle to that of normal ones. Results: The diploid number of metaphase chromosomes in arsenic affected cattle as well as in normal cattle were all 2n=60, 58 being autosomes and 2 being sex chromosomes. From the centromeric position, karyotyping studies revealed that all the 29 pair of autosomes was found to be acrocentric or telocentric, and the sex chromosomes (XX were submetacentric in both normal and arsenic affected cattle. The relative length of all the autosome pairs and sex chrosomosome pair was found to be higher in normal than that of arsenic affected cattle. The mean arm ratio of X-chromosome was higher in normal than that of arsenic affected cattle, but it is reverse in case of centromere index value of X-chromosome. There was no significant difference of arm ratio and centromere index of X-chromosomes between arsenic affected and normal cattle. No chromosomal abnormalities were found in arsenic affected cattle. Conclusion: The chromosome analysis of arsenic affected cattle in West Bengal reported for the first time in this present study which may serve as a guideline for future studies in other species. These reference values will also help in comparison of cytological studies of arsenic affected cattle to that of various toxicants.

  6. The Dark Side of the Affective Profiles

    Directory of Open Access Journals (Sweden)

    Danilo Garcia

    2015-12-01

    Full Text Available The affective profiles model is based on the combination of individuals’ experience of high/low positive affect and high/low negative affect: self-fulfilling, high affective, low affective, and self-destructive. We used the profiles as the backdrop for the investigation of individual differences in malevolent character traits (i.e., the Dark Triad: psychopathy, Machiavellianism, and narcissism. A total of 1,000 participants (age: M = 31.50 SD = 10.27, 667 males and 333 females, recruited through Amazons’ Mechanical Turk (MTurk, responded to the Positive Affect Negative Affect Schedule and the Dark Triad Dirty Dozen. Individuals with a high affective profile reported higher degree of narcissism than those with any other profile, and together with individuals with a self-destructive profile, also higher degree of Machiavellianism and psychopathy than individuals with a low affective and self-fulfilling profile. Males scored higher in Machiavellianism and psychopathy. Together with earlier findings, our results show that while individuals in both the self-fulfilling and high affective profiles are extrovert and self-directed, only those in the high affective profile express an immature and malevolent character (i.e., high levels of all Dark Triad traits. Conversely, individuals in the self-fulfilling profile have earlier reported higher levels of cooperativeness and faith. More importantly, the unique association between high levels of positive emotions and narcissism and the unified association between negative emotions to both psychopathy and Machiavellianism imply a dyad rather than a triad of malevolent character traits.

  7. Affect Recognition in Adults with ADHD

    Science.gov (United States)

    Miller, Meghan; Hanford, Russell B.; Fassbender, Catherine; Duke, Marshall; Schweitzer, Julie B.

    2011-01-01

    Objective: This study compared affect recognition abilities between adults with and without ADHD. Method: The sample consisted of 51 participants (34 men, 17 women) divided into 3 groups: ADHD-combined type (ADHD-C; n = 17), ADHD-predominantly inattentive type (ADHD-I; n = 16), and controls (n = 18). The mean age was 34 years. Affect recognition…

  8. Genetic search feature selection for affective modeling

    DEFF Research Database (Denmark)

    Martínez, Héctor P.; Yannakakis, Georgios N.

    2010-01-01

    Automatic feature selection is a critical step towards the generation of successful computational models of affect. This paper presents a genetic search-based feature selection method which is developed as a global-search algorithm for improving the accuracy of the affective models built...

  9. Affective Atmospheres in the House of Usher

    DEFF Research Database (Denmark)

    Brink, Dennis Meyhoff

    2016-01-01

    Emotional intensities do not only pertain to the ‘inner life’ of individuals; they are also to be found, as the saying goes, ‘in the air,’ i.e. as shared atmospheres that envelope and affect us. Such affective atmospheres are omnipresent in Edgar Allan Poe’s short story “The Fall of the House of ...

  10. Mental and physical effort affect vigilance differently

    NARCIS (Netherlands)

    Smit, A.S.; Eling, P.A.T.M.; Hopman, M.T.E.; Coenen, A.M.L.

    2005-01-01

    Both physical and mental effort are thought to affect vigilance. Mental effort is known for its vigilance declining effects, but the effects of physical effort are less clear. This study investigated whether these two forms of effort affect the EEG and subjective alertness differently. Participants

  11. Mental and physical effort affect vigilance differently.

    NARCIS (Netherlands)

    Smit, A.S.; Eling, P.A.T.M.; Hopman, M.T.E.; Coenen, A.M.L.

    2005-01-01

    Both physical and mental effort are thought to affect vigilance. Mental effort is known for its vigilance declining effects, but the effects of physical effort are less clear. This study investigated whether these two forms of effort affect the EEG and subjective alertness differently. Participants

  12. Nonverbal synchrony and affect in dyadic interactions

    Directory of Open Access Journals (Sweden)

    Wolfgang eTschacher

    2014-11-01

    Full Text Available In an experiment on dyadic social interaction, we invited participants to verbal interactions in cooperative, competitive, and 'fun task' conditions. We focused on the link between interactants' affectivity and their nonverbal synchrony, and explored which further variables contributed to affectivity: interactants' personality traits, sex, and the prescribed interaction tasks. Nonverbal synchrony was quantified by the coordination of interactants' body movement, using an automated video-analysis algorithm (Motion Energy Analysis, MEA. Traits were assessed with standard questionnaires of personality, attachment, interactional style, psychopathology and interpersonal reactivity. We included 168 previously unacquainted individuals who were randomly allocated to same-sex dyads (84 females, 84 males, mean age 27.3 years. Dyads discussed four topics of general interest drawn from an urn of eight topics, and finally engaged in a fun interaction. Each interaction lasted five minutes. In between interactions, participants repeatedly assessed their affect. Using hierarchical linear modeling, we found moderate to strong effect sizes for synchrony to occur, especially in competitive and fun task conditions. Positive affect was associated positively with synchrony, negative affect was associated negatively. As for causal direction, data supported the interpretation that synchrony entailed affect rather than vice versa. The link between nonverbal synchrony and affect was strongest in female dyads. The findings extend previous reports of synchrony and mimicry associated with emotion in relationships and suggest a possible mechanism of the synchrony-affect correlation.

  13. How Does Climate Change Affect Biodiversity?

    DEFF Research Database (Denmark)

    Bastos Araujo, Miguel; Rahbek, Carsten

    2006-01-01

    The most recent and complex bioclimate models excel at describing species' current distributions. Yet, it is unclear which models will best predict how climate change will affect their future distributions.......The most recent and complex bioclimate models excel at describing species' current distributions. Yet, it is unclear which models will best predict how climate change will affect their future distributions....

  14. Mental health in war-affected populations

    NARCIS (Netherlands)

    Scholte, W.F.

    2013-01-01

    This book addresses mental health problems in populations in nonwestern war-affected regions, and methods to mitigate these problems through interventions focusing on social reintegration. It describes a number of studies among war-affected populations in widely different areas: refugees from the Rw

  15. 40 CFR 1502.15 - Affected environment.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Affected environment. 1502.15 Section 1502.15 Protection of Environment COUNCIL ON ENVIRONMENTAL QUALITY ENVIRONMENTAL IMPACT STATEMENT § 1502.15 Affected environment. The environmental impact statement shall succinctly describe...

  16. Did Johnson affect Trump or Clinton?

    DEFF Research Database (Denmark)

    Kurrild-Klitgaard, Peter

    ’s candidacy affected both Hillary Clinton and Donald Trump, did so moderately, and none in particular. When controlling for other factors Clinton’s lead seems to have been negatively affected. This suggests that Libertarian support in 2016 to a large degree has come from voters who otherwise would have split...

  17. Heritability of bipolar affective disorder: Family study

    Directory of Open Access Journals (Sweden)

    Obradović Tanja

    2011-01-01

    Full Text Available Background/Aim. Bipolar affective disorder is mental disorder with polygenic type of heredity. Heritability - relation between genetic and environmental variance is used to estimate the level of influence of genetic variance to phenotype variance. Study results show decreasing trend in the value of heritability of bipolar affective disorder, thus indicating that this disorder is a complex behavioral threshold characteristic. Therefore, the aim of this study was to estimate the contribution of genetic variance to phenotype variance of bipolar affective disorder, i.e. to estimate heritability of this disorder. Methods. By the use of a questionnaire, 80 patients with over crossed threshold for bipolar affective disorder were asked for functional information about the members of their families belonging to the first degree of relation (fathers, mothers and full- sibs. By using ”Applet for calculating heritability for threshold traits (disease“, and regression analysis, heritability of bipolar affective disorder as well as its statistical significance, were estimated (χ2 test. Results. Heritability and relationship of genetic and environmental variance of bipolar affective disorder is 0.2 with statistically significant difference from zero (p < 0.001. Conclusion. The estimated contribution of genetic variance to phenotype variance of bipolar affective disorder is low being 20%, while the contribution of environmental variance is 80%. This result contributes to the understanding of bipolar affective disorder as a complex behavioral threshold trait.

  18. Trait Affect and Job Search Outcomes

    Science.gov (United States)

    Cote, Stephane; Saks, Alan M.; Zikic, Jelena

    2006-01-01

    The present study examines the role of trait affect in job search. One hundred and twenty-three university students completed measures of positive and negative affectivity, conscientiousness, job search self-efficacy, job search clarity, and job search intensity during their last year of school while on the job market. At the end of the school…

  19. Affective Brain-Computer Interfaces: Preface

    NARCIS (Netherlands)

    Mühl, Christian; Heylen, Dirk; Nijholt, Anton; Mühl, Christian; Heylen, Dirk; Nijholt, Anton

    2009-01-01

    These are the proceedings of ABCI 2009, Affective Brain Computer Interfaces, a workshop that was organized in conjunction with ACII 2009, the International Conference on Affective Computation and Intelligent Interaction, held in Amsterdam, The Netherlands, September 2009. The workshop took place on

  20. Exploiting facial expressions for affective video summarisation

    NARCIS (Netherlands)

    Joho, H.; Jose, J.M.; Valenti, R.; Sebe, N.; Marchand-Maillet, S.; Kompatsiaris, I.

    2009-01-01

    This paper presents an approach to affective video summarisation based on the facial expressions (FX) of viewers. A facial expression recognition system was deployed to capture a viewer's face and his/her expressions. The user's facial expressions were analysed to infer personalised affective scenes

  1. Affective Priming with Auditory Speech Stimuli

    Science.gov (United States)

    Degner, Juliane

    2011-01-01

    Four experiments explored the applicability of auditory stimulus presentation in affective priming tasks. In Experiment 1, it was found that standard affective priming effects occur when prime and target words are presented simultaneously via headphones similar to a dichotic listening procedure. In Experiment 2, stimulus onset asynchrony (SOA) was…

  2. Affective priming with auditory speech stimuli

    NARCIS (Netherlands)

    Degner, J.

    2011-01-01

    Four experiments explored the applicability of auditory stimulus presentation in affective priming tasks. In Experiment 1, it was found that standard affective priming effects occur when prime and target words are presented simultaneously via headphones similar to a dichotic listening procedure. In

  3. Affective Understanding and the Reading of Poetry.

    Science.gov (United States)

    Vine, Harold A., Jr.

    This investigation was designed to shed light on the study of literature by focusing on the reader's affective understandings and by using the semantic differential (S.D.) to measure affective meaning. Initially, an experimental group of 49 advanced senior high school students and a comparison group of 53 average senior high students used the S.D.…

  4. Affective multimodal mirror: sensing and eliciting laughter

    NARCIS (Netherlands)

    Melder, W.A.; Truong, K.P.; Uyl, M. den; Leeuwen, D.A. van; Neerincx, M.A.; Loos, L.R.; Stock Plum, B.

    2007-01-01

    In this paper, we present a multimodal affective mirror that senses and elicits laughter. Currently, the mirror contains a vocal and a facial affect-sensing module, a component that fuses the output of these two modules to achieve a user-state assessment, a user state transition model, and a compone

  5. Leader affective presence and innovation in teams.

    Science.gov (United States)

    Madrid, Hector P; Totterdell, Peter; Niven, Karen; Barros, Eduardo

    2016-05-01

    Affective presence is a novel personality construct that describes the tendency of individuals to make their interaction partners feel similarly positive or negative. We adopt this construct, together with the input-process-output model of teamwork, to understand how team leaders influence team interaction and innovation performance. In 2 multisource studies, based on 350 individuals working in 87 teams of 2 public organizations and 734 individuals working in 69 teams of a private organization, we tested and supported hypotheses that team leader positive affective presence was positively related to team information sharing, whereas team leader negative affective presence was negatively related to the same team process. In turn, team information sharing was positively related to team innovation, mediating the effects of leader affective presence on this team output. The results indicate the value of adopting an interpersonal individual differences approach to understanding how affect-related characteristics of leaders influence interaction processes and complex performance in teams. (PsycINFO Database Record

  6. Affect and Public Support for Military Action

    Directory of Open Access Journals (Sweden)

    Dukhong Kim

    2014-12-01

    Full Text Available This study examines the effects of affect on public opinion on foreign policy. It extends the existing studies which show a significant role that affect, as measured by feelings toward a country, plays in shaping public opinion on military action. According to the existing theory, the mass public, which does not have high levels of political information and knowledge, can rely on affect to make reasonable decisions and opinions. This is possible because affect works as an information shortcut or heuristic that can help those individuals who lack cognitive capacity to engage in a systematic search for information and a decision-making process. The research finding confirms this theory. More importantly, this study extends the existing studies by elaborating the conditions under which affect works in accounting for individuals’ support for military intervention. The effect of affect is conditioned by the level of political knowledge, which shows that knowledgeable individuals are more adept at using affect as a heuristic tool.

  7. Embodied affectivity: On moving and being moved

    Directory of Open Access Journals (Sweden)

    Thomas eFuchs

    2014-06-01

    Full Text Available There is a growing body of research indicating that bodily sensation and behaviour strongly influences one’s emotional reaction towards certain situations or objects. On this background, a framework model of embodied affectivity is suggested: we regard emotions as resulting from the circular interaction between affective qualities or affordances in the environment and the subject’s bodily resonance, be it in the form of sensations, postures, expressive movements or movement tendencies. Motion and emotion are thus intrinsically connected: one is moved by movement (perception; impression; affection and moved to move (action; expression; e-motion. Through its resonance, the body functions as a medium of emotional perception: it colours or charges self-experience and the environment with affective valences while it remains itself in the background of one’s own awareness. This model is then applied to emotional social understanding or interaffectivity which is regarded as an intertwinement of two cycles of embodied affectivity, thus continuously modifying each partner’s affective affordances and bodily resonance. We conclude with considerations of how embodied affectivity is altered in psychopathology and can be addressed in psychotherapy of the embodied self.

  8. Affective brain-computer music interfacing

    Science.gov (United States)

    Daly, Ian; Williams, Duncan; Kirke, Alexis; Weaver, James; Malik, Asad; Hwang, Faustina; Miranda, Eduardo; Nasuto, Slawomir J.

    2016-08-01

    Objective. We aim to develop and evaluate an affective brain-computer music interface (aBCMI) for modulating the affective states of its users. Approach. An aBCMI is constructed to detect a user's current affective state and attempt to modulate it in order to achieve specific objectives (for example, making the user calmer or happier) by playing music which is generated according to a specific affective target by an algorithmic music composition system and a case-based reasoning system. The system is trained and tested in a longitudinal study on a population of eight healthy participants, with each participant returning for multiple sessions. Main results. The final online aBCMI is able to detect its users current affective states with classification accuracies of up to 65% (3 class, p\\lt 0.01) and modulate its user's affective states significantly above chance level (p\\lt 0.05). Significance. Our system represents one of the first demonstrations of an online aBCMI that is able to accurately detect and respond to user's affective states. Possible applications include use in music therapy and entertainment.

  9. Implicit processing of visual emotions is affected by sound-induced affective states and individual affective traits.

    Directory of Open Access Journals (Sweden)

    Tiziana Quarto

    Full Text Available The ability to recognize emotions contained in facial expressions are affected by both affective traits and states and varies widely between individuals. While affective traits are stable in time, affective states can be regulated more rapidly by environmental stimuli, such as music, that indirectly modulate the brain state. Here, we tested whether a relaxing or irritating sound environment affects implicit processing of facial expressions. Moreover, we investigated whether and how individual traits of anxiety and emotional control interact with this process. 32 healthy subjects performed an implicit emotion processing task (presented to subjects as a gender discrimination task while the sound environment was defined either by a a therapeutic music sequence (MusiCure, b a noise sequence or c silence. Individual changes in mood were sampled before and after the task by a computerized questionnaire. Additionally, emotional control and trait anxiety were assessed in a separate session by paper and pencil questionnaires. Results showed a better mood after the MusiCure condition compared with the other experimental conditions and faster responses to happy faces during MusiCure compared with angry faces during Noise. Moreover, individuals with higher trait anxiety were faster in performing the implicit emotion processing task during MusiCure compared with Silence. These findings suggest that sound-induced affective states are associated with differential responses to angry and happy emotional faces at an implicit stage of processing, and that a relaxing sound environment facilitates the implicit emotional processing in anxious individuals.

  10. Affectivity in the Young Sartre's Intersubjective Theory

    Directory of Open Access Journals (Sweden)

    Carlos de la Puente

    2006-12-01

    Full Text Available The intersubjective theory that Sartre proposed in Being and Nothingness contends that human encounters are necessarily reifying. The author proposes that one of the constitutive theoretical elements of this pessimistic view of human encounters is Sartre’s conception of affectivity as a degradation of consciousness. The author explores this vision of affection that Sartre initially developed in his essay Outline of a Theory of Emotions, and concludes that it was this mode of understanding affectivity that later decisively influenced his contention in Being and Nothingness that the essence of human relations is conflict.

  11. Introduction: Analysing Emotion and Theorising Affect

    Directory of Open Access Journals (Sweden)

    Peta Tait

    2016-08-01

    Full Text Available This discussion introduces ideas of emotion and affect for a volume of articles demonstrating the scope of approaches used in their study within the humanities and creative arts. The volume offers multiple perspectives on emotion and affect within 20th-century and 21st-century texts, arts and organisations and their histories. The discussion explains how emotion encompasses the emotions, emotional feeling, sensation and mood and how these can be analysed particularly in relation to literature, art and performance. It briefly summarises concepts of affect theory within recent approaches before introducing the articles.

  12. How Does Heart Disease Affect Women?

    Science.gov (United States)

    ... page from the NHLBI on Twitter. How Does Heart Disease Affect Women? Español In the United States, 1 ... about coronary MVD and broken heart syndrome. Coronary Heart Disease CHD is a disease in which plaque (plak) ...

  13. Heart Disease Affects Women of All Ages

    Science.gov (United States)

    Skip Navigation Bar Home Current Issue Past Issues Heart Disease Affects Women of All Ages Past Issues / Winter ... weeks of a heart attack. For Women with Heart Disease: About 6 million American women have coronary heart ...

  14. Alcohol: Does It Affect Blood Pressure?

    Science.gov (United States)

    ... and Conditions High blood pressure (hypertension) Does drinking alcohol affect your blood pressure? Answers from Sheldon G. Sheps, M.D. Drinking too much alcohol can raise blood pressure to unhealthy levels. Having ...

  15. Embedding Affective Learning Outcomes in Library Instruction

    Directory of Open Access Journals (Sweden)

    Ellysa Stern Cahoy

    2012-12-01

    Full Text Available While information literacy in higher education has long been focused on cognitive learning outcomes, attention must be paid to students’ affective, emotional needs throughout the research process. This article identifies models for embedding affective learning outcomes within information literacy instruction, and provides strategies to help librarians discover, articulate, and address students’ self-efficacy, motivation, emotions and attitudes. Worksheets to assist in creating affective learning outcomes are included to bring structure to an area of learning that is often challenging to articulate and measure. Also included in the article are the results of a recent survey of instruction librarians’ familiarity and inclusion of affective learning outcomes within teaching and learning initiatives.

  16. Sperm Shape (Morphology): Does It Affect Fertility?

    Science.gov (United States)

    ... Website of the American Society for Reproductive Medicine Sperm morphology (shape): Does it affect fertility? How is ... semen analysis. This measures many features of the sperm and semen (the fluid in which the sperm ...

  17. 28 CFR 55.15 - Affected activities.

    Science.gov (United States)

    2010-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively...

  18. Organizational behavior: affect in the workplace.

    Science.gov (United States)

    Brief, Arthur P; Weiss, Howard M

    2002-01-01

    The study of affect in the workplace began and peaked in the 1930s, with the decades that followed up to the 1990s not being particularly fertile. Whereas job satisfaction generally continues to be loosely but not carefully thought of and measured as an affective state, critical work in the 1990s has raised serious questions about the affective status of job satisfaction in terms of its causes as well as its definition and measurement. Recent research has focused on the production of moods and emotions at work, with an emphasis, at least conceptually, on stressful events, leaders, work groups, physical settings, and rewards/punishment. Other recent research has addressed the consequences of workers' feelings, in particular, a variety of performance outcomes (e.g., helping behaviors and creativity). Even though recent interest in affect in the workplace has been intense, many theoretical and methodological opportunities and challenges remain.

  19. How Will Cancer Affect My Sex Life?

    Science.gov (United States)

    ... Patients and Families How will cancer affect my sex life? Sexual feelings and attitudes vary greatly among ... Others find that they have less interest in sex because of the physical and emotional demands of ...

  20. Environmental factors affecting autoimmune thyroid disease

    Energy Technology Data Exchange (ETDEWEB)

    Safran, M.; Paul, T.L.; Roti, E.; Braverman, L.E.

    1987-06-01

    A number of environmental factors affect the incidence and progression of autoimmune thyroid disease. Exposure to excess iodine, certain drugs, infectious agents and pollutants, and stress have all been implicated.

  1. Certain Bacteria May Affect Preterm Birth Risk

    Science.gov (United States)

    ... https://medlineplus.gov/news/fullstory_163401.html Certain Bacteria May Affect Preterm Birth Risk Bad 'bugs' tied ... Feb. 3, 2017 (HealthDay News) -- Certain types of bacteria in a pregnant woman's cervix and vagina can ...

  2. Mental Health May Affect Chances Against Cancer

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_163241.html Mental Health May Affect Chances Against Cancer Early research suggests ... Our findings contribute to the evidence that poor mental health might have some predictive capacity for certain physical ...

  3. Affective intentionality and self-consciousness.

    Science.gov (United States)

    Slaby, Jan; Stephan, Achim

    2008-06-01

    We elaborate and defend the claim that human affective states ("feelings") are, among other things, self-disclosing. We will show why affective intentionality has to be considered in order to understand human self-consciousness. One specific class of affective states, so-called existential feelings, although often neglected in philosophical treatments of emotions, will prove central. These feelings importantly pre-structure affective and other intentional relations to the world. Our main thesis is that existential feelings are an important manifestation of self-consciousness and figure prominently in human self-understanding. We offer an ordering of four levels of existential feelings and also give considerations in favour of the essential bodily nature of these feelings.

  4. Postpartum Depression Affects New Dads, Too

    Science.gov (United States)

    ... https://medlineplus.gov/news/fullstory_163092.html Postpartum Depression Affects New Dads, Too Certain men at greater ... HealthDay News) -- Men can also suffer from postpartum depression after their baby is born. "Dads want to ...

  5. Will Stress during Pregnancy Affect My Baby?

    Science.gov (United States)

    ... drinking, which contribute to other problems. 3 Reducing stress is important for preventing problems during your pregnancy and for reducing your risk for health problems that may affect your developing child. Identify the source of your ...

  6. ENVIRONMENTAL FACTORS AFFECTING BREAST CANCER SUSCEPTIBILITY

    Science.gov (United States)

    Environmental Factors Affecting Breast Cancer SusceptibilitySuzanne. E. FentonUS EPA, ORD, MD-67 NHEERL, Reproductive Toxicology Division, Research Triangle Park, NC 27711.Breast cancer is still the most common malignancy afflicting women in the Western world. Alt...

  7. Working night shifts affects surgeons' biological rhythm

    DEFF Research Database (Denmark)

    Amirian, Ilda; Andersen, Lærke T; Rosenberg, Jacob

    2015-01-01

    BACKGROUND: Chronic sleep deprivation combined with work during the night is known to affect performance and compromise residents' own safety. The aim of this study was to examine markers of circadian rhythm and the sleep-wake cycle in surgeons working night shifts. METHODS: Surgeons were monitored...... and on PC1. For all subjective measures, a marked deterioration was seen on PC1. CONCLUSION: Surgeons' circadian rhythm was affected by working night shifts....

  8. Nurse Educator’s Affective Teaching Strategies

    Directory of Open Access Journals (Sweden)

    Myra C. Britiller

    2014-02-01

    Full Text Available Teaching is highly charged with feeling, aroused by and directed towards not just people, but also values and ideals (Garritz, 2010. Emotions and feelings greatly affect the way the students learn and acquire knowledge. There must be a balance between the three domains of learning because once one domain is left behind, the other domains will be affected. The purpose of this study is to determine nurse educators’ affective teaching strategies. Specifically, it assessed the strategies of nurse educators on how to develop the affective domain of student nurses. In addition, the study assessed the values of student nurses in terms of God-centeredness, Leadership, Integrity, and Nationalism. Lastly, the researchers proposed innovative teaching methodologies to enhance the attitudes of nursing students. This study used descriptive research design. The respondents of the study were the nursing students and clinical instructors of Lyceum of the Philippines University – Batangas. 190 student nurses were chosen through stratified random sampling while 10 clinical instructors were chosen based on the number of times they handled the students both in lecture and clinical area. The instruments were selfmade and standardized questionnaire and interview guide to obtain the data needed for the study. The data gathered are compiled, organized and tabulated for statistical treatment. Weighted mean was utilized to compute the quantitative data whereas the interview was used to form the qualitative analysis. Role modelling is the most common strategy of nurse educators to enhance the affective domain of student nurses. Their actions consciously and unconsciously affecting the attitude of the students. Hence, confidence among the students needs most attention in developing affective domain as it produce change in other values presented in the study. The proposed teaching strategies aim to enhance the affective domain of nursing students in line with the

  9. Fight or flight: Affective news framing effects

    OpenAIRE

    Feinholdt, A.

    2016-01-01

    This dissertation investigates how emotions shape and are shaped by news framing and how this interplay leads to a number of affective news framing effects. My work reveals that emotions do not only function as mechanisms but also as channels strengthening or weakening framing effects. In addition, I show that issue characteristics and the selected news frame can determine whether and how emotions are elicited. Further, I provide evidence that affective news framing effects can arise as the r...

  10. Factors Affecting Students’ Achievement in Mathematics ∗

    OpenAIRE

    2010-01-01

    In this study, the factors that affect students’ mathematics achievement in secondary school mathematics grades of 6th, 7th and 8th were explored. For this purpose, the factors that affect mathematics education and mathematics achievement such as type of school, family income, studying time and students’ attitude towards mathematics and attendance to private courses have been investigated. The sample of the study consisted of 275 students from one private (n=58) and two state schools (n= 217)...

  11. Heritability of bipolar affective disorder: Family study

    OpenAIRE

    Obradović Tanja; Veličković Ružica; Timotijević Ivana; Anđelković Marko

    2011-01-01

    Background/Aim. Bipolar affective disorder is mental disorder with polygenic type of heredity. Heritability - relation between genetic and environmental variance is used to estimate the level of influence of genetic variance to phenotype variance. Study results show decreasing trend in the value of heritability of bipolar affective disorder, thus indicating that this disorder is a complex behavioral threshold characteristic. Therefore, the aim of this study was to estimate the contribut...

  12. Go Naked: Diapers Affect Infant Walking

    OpenAIRE

    Cole, Whitney G.; Lingeman, Jesse M.; Adolph, Karen E.

    2012-01-01

    In light of cross-cultural and experimental research highlighting effects of childrearing practices on infant motor skill, we asked whether wearing diapers, a seemingly innocuous childrearing practice, affects infant walking. Diapers introduce bulk between the legs, potentially exacerbating infants’ poor balance and wide stance. We show that walking is adversely affected by old-fashioned cloth diapers, and that even modern disposable diapers—habitually worn by most infants in the sample—incur...

  13. Overcoming Affective Barriers in College English Learning

    Institute of Scientific and Technical Information of China (English)

    任永东

    2014-01-01

    Nowadays, the development of economy as well as the progress of society requires better English competence of col-lege students. As a result, a large number of college students are obsessedby affective barriers in their English learning, especially when they have to cope with CET 4 or CET 6, which is more and more difficult. This paper focuses on how to help college stu-dents overcome their affective barriers more effectively to improve their English learning.

  14. Stability of Facial Affective Expressions in Schizophrenia

    Directory of Open Access Journals (Sweden)

    H. Fatouros-Bergman

    2012-01-01

    Full Text Available Thirty-two videorecorded interviews were conducted by two interviewers with eight patients diagnosed with schizophrenia. Each patient was interviewed four times: three weekly interviews by the first interviewer and one additional interview by the second interviewer. 64 selected sequences where the patients were speaking about psychotic experiences were scored for facial affective behaviour with Emotion Facial Action Coding System (EMFACS. In accordance with previous research, the results show that patients diagnosed with schizophrenia express negative facial affectivity. Facial affective behaviour seems not to be dependent on temporality, since within-subjects ANOVA revealed no substantial changes in the amount of affects displayed across the weekly interview occasions. Whereas previous findings found contempt to be the most frequent affect in patients, in the present material disgust was as common, but depended on the interviewer. The results suggest that facial affectivity in these patients is primarily dominated by the negative emotions of disgust and, to a lesser extent, contempt and implies that this seems to be a fairly stable feature.

  15. Dynamic Musical Communication of Core Affect

    Directory of Open Access Journals (Sweden)

    Nicole eFlaig

    2014-03-01

    Full Text Available Is there something special about the way music communicates feelings? Theorists since Meyer (1956 have attempted to explain how music could stimulate varied and subtle affective experiences by violating learned expectancies, or by mimicking other forms of social interaction. Our proposal is that music speaks to the brain in its own language; it need not imitate any other form of communication. We review recent theoretical and empirical literature, which suggests that all conscious processes consist of dynamic neural events, produced by spatially dispersed processes in the physical brain. Intentional thought and affective experience arise as dynamical aspects of neural events taking place in multiple brain areas simultaneously. At any given moment, this content comprises a unified scene that is integrated into a dynamic core through synchrony of neuronal oscillations. We propose that 1 neurodynamic synchrony with musical stimuli gives rise to musical qualia including tonal and temporal expectancies, and that 2 music-synchronous responses couple into core neurodynamics, enabling music to directly modulate core affect. Expressive music performance, for example, may recruit rhythm-synchronous neural responses to support affective communication. We suggest that the dynamic relationship between musical expression and the experience of affect presents a unique opportunity for the study of emotional experience. This may help elucidate the neural mechanisms underlying arousal and valence, and offer a new approach to exploring the complex dynamics of the how and why of emotional experience.

  16. Dynamic musical communication of core affect.

    Science.gov (United States)

    Flaig, Nicole K; Large, Edward W

    2014-01-01

    Is there something special about the way music communicates feelings? Theorists since Meyer (1956) have attempted to explain how music could stimulate varied and subtle affective experiences by violating learned expectancies, or by mimicking other forms of social interaction. Our proposal is that music speaks to the brain in its own language; it need not imitate any other form of communication. We review recent theoretical and empirical literature, which suggests that all conscious processes consist of dynamic neural events, produced by spatially dispersed processes in the physical brain. Intentional thought and affective experience arise as dynamical aspects of neural events taking place in multiple brain areas simultaneously. At any given moment, this content comprises a unified "scene" that is integrated into a dynamic core through synchrony of neuronal oscillations. We propose that (1) neurodynamic synchrony with musical stimuli gives rise to musical qualia including tonal and temporal expectancies, and that (2) music-synchronous responses couple into core neurodynamics, enabling music to directly modulate core affect. Expressive music performance, for example, may recruit rhythm-synchronous neural responses to support affective communication. We suggest that the dynamic relationship between musical expression and the experience of affect presents a unique opportunity for the study of emotional experience. This may help elucidate the neural mechanisms underlying arousal and valence, and offer a new approach to exploring the complex dynamics of the how and why of emotional experience.

  17. Recent developments in affective recommender systems

    Science.gov (United States)

    Katarya, Rahul; Verma, Om Prakash

    2016-11-01

    Recommender systems (RSs) are playing a significant role since 1990s as they provide relevant, personalized information to the users over the internet. Lots of work have been done in information filtering, utilization, and application related to RS. However, an important area recently draws our attention which is affective recommender system. Affective recommender system (ARS) is latest trending area of research, as publication in this domain are few and recently published. ARS is associated with human behaviour, human factors, mood, senses, emotions, facial expressions, body gesture and physiological with human-computer interaction (HCI). Due to this assortment and various interests, more explanation is required, as it is in premature phase and growing as compared to other fields. So we have done literature review (LR) in the affective recommender systems by doing classification, incorporate reputed articles published from the year 2003 to February 2016. We include articles which highlight, analyse, and perform a study on affective recommender systems. This article categorizes, synthesizes, and discusses the research and development in ARS. We have classified and managed ARS papers according to different perspectives: research gaps, nature, algorithm or method adopted, datasets, the platform on executed, types of information and evaluation techniques applied. The researchers and professionals will positively support this survey article for understanding the current position, research in affective recommender systems and will guide future trends, opportunity and research focus in ARS.

  18. Positive Affect and Negative Affect as Modulators of Cognition and Motivation: The Rediscovery of Affect in Achievement Goal Theory

    Science.gov (United States)

    Bjornebekk, Gunnar

    2008-01-01

    A central hypothesis of classical motivation theory is that affect underlies motivation and its behavioural manifestations. However, this has been largely ignored in the past 30 years because social cognitivism has been the dominant theory. As a result, studies have concentrated on social cognitive processes when analysing those factors that…

  19. Neuromodulation, Emotional Feelings and Affective Disorders

    Directory of Open Access Journals (Sweden)

    Fushun Wang

    2016-01-01

    Full Text Available Affective disorders such as anxiety, phobia and depression are a leading cause of disabilities worldwide. Monoamine neuromodulators are used to treat most of them, with variable degrees of efficacy. Here, we review and interpret experimental findings about the relation of neuromodulation and emotional feelings, in pursuit of two goals: (a to improve the conceptualisation of affective/emotional states, and (b to develop a descriptive model of basic emotional feelings related to the actions of neuromodulators. In this model, we hypothesize that specific neuromodulators are effective for basic emotions. The model can be helpful for mental health professionals to better understand the affective dynamics of persons and the actions of neuromodulators - and respective psychoactive drugs - on this dynamics.

  20. Affective signals of threat increase perceived proximity.

    Science.gov (United States)

    Cole, Shana; Balcetis, Emily; Dunning, David

    2013-01-01

    Do stimuli appear to be closer when they are more threatening? We tested people's perceptions of distance to stimuli that they felt were threatening relative to perceptions of stimuli they felt were disgusting or neutral. Two studies demonstrated that stimuli that emitted affective signals of threat (e.g., an aggressive male student) were seen as physically closer than stimuli that emitted affective signals of disgust (e.g., a repulsive male student) or no affective signal. Even after controlling for the direct effects of physiological arousal, object familiarity, and intensity of the negative emotional reaction, we found that threatening stimuli appeared to be physically closer than did disgusting ones (Study 2). These findings highlight the links among biased perception, action regulation, and successful navigation of the environment.

  1. Naivete and need for affection among pedophiles.

    Science.gov (United States)

    Johnston, S A; French, A P; Schouweiler, W F; Johnston, F A

    1992-09-01

    MMPI scores for items on the Harris and Lingoes (1955, 1968) subscales HY2 (need for affection) and PA2 (naivete) were compared among pedophiles (n = 50), non-sexually deviant psychiatric patients (n = 25), and a general control group (n = 50). Results indicated that pedophiles did not demonstrate a "naive need for affection," but, rather, a cynical need for affection. Scores on the Pedophelia (PE) Scale (Toobert, Bartelme, & Jones, 1959) also were compared, and no significant differences were found between the pedophiles and either control group, although a difference was found between the scores of the psychiatric control group and those of the general sample. Selected items from scale 4 (Psychopathic Deviate) and scale 9 (Hypomania) also produced no significant differences among groups.

  2. Do recreational activities affect coastal biodiversity?

    Science.gov (United States)

    Riera, Rodrigo; Menci, Cristiano; Sanabria-Fernández, José Antonio; Becerro, Mikel A.

    2016-09-01

    Human activities are largely affecting coastal communities worldwide. Recreational perturbations have been overlooked in comparison to other perturbations, yet they are potential threats to marine biodiversity. They affect coastal communities in different ways, underpinning consistent shifts in fish and invertebrates assemblages. Several sites were sampled subjected to varying effects by recreational fishermen (low and high pressure) and scuba divers (low and high) in an overpopulated Atlantic island. Non-consistent differences in ecological, trophic and functional diversity were found in coastal communities, considering both factors ("diving" and "fishing"). Multivariate analyses only showed significant differences in benthic invertebrates between intensively-dived and non-dived sites. The lack of clear trends may be explained by the depletion of coastal resources in the study area, an extensively-affected island by overfishing.

  3. Factors Affecting University Library Website Design

    Directory of Open Access Journals (Sweden)

    Yongi-Mi Kim

    2011-09-01

    Full Text Available Existing studies have extensively explored factors that affect users’ intentions to use university library website resources (ULWR; yet little attention has been given to factors affecting university library website design. This paper investigates factors that affect university library website design and assesses the success of the university library website from both designers’ and users’ perspectives. The findings show that when planning a website, university web designers consider university guidelines, review other websites, and consult with experts and other divisions within the library; however, resources and training for the design process are lacking. While website designers assess their websites as highly successful, user evaluations are somewhat lower. Accordingly, use is low, and users rely heavily on commercial websites. Suggestions for enhancing the usage of ULWR are provided.

  4. Affective processing in bilingual speakers: disembodied cognition?

    Science.gov (United States)

    Pavlenko, Aneta

    2012-01-01

    A recent study by Keysar, Hayakawa, and An (2012) suggests that "thinking in a foreign language" may reduce decision biases because a foreign language provides a greater emotional distance than a native tongue. The possibility of such "disembodied" cognition is of great interest for theories of affect and cognition and for many other areas of psychological theory and practice, from clinical and forensic psychology to marketing, but first this claim needs to be properly evaluated. The purpose of this review is to examine the findings of clinical, introspective, cognitive, psychophysiological, and neuroimaging studies of affective processing in bilingual speakers in order to identify converging patterns of results, to evaluate the claim about "disembodied cognition," and to outline directions for future inquiry. The findings to date reveal two interrelated processing effects. First-language (L1) advantage refers to increased automaticity of affective processing in the L1 and heightened electrodermal reactivity to L1 emotion-laden words. Second-language (L2) advantage refers to decreased automaticity of affective processing in the L2, which reduces interference effects and lowers electrodermal reactivity to negative emotional stimuli. The differences in L1 and L2 affective processing suggest that in some bilingual speakers, in particular late bilinguals and foreign language users, respective languages may be differentially embodied, with the later learned language processed semantically but not affectively. This difference accounts for the reduction of framing biases in L2 processing in the study by Keysar et al. (2012). The follow-up discussion identifies the limits of the findings to date in terms of participant populations, levels of processing, and types of stimuli, puts forth alternative explanations of the documented effects, and articulates predictions to be tested in future research.

  5. ON AFFECTIVE BARRIERS TO LANGUAGE LEARNING

    Institute of Scientific and Technical Information of China (English)

    XiangMaoying

    2004-01-01

    Affective factors play a significant role in languagelearning. This paper argues that positive emotions can facilitatethe language learning process and improve learners' languageperformance, while negative emotions will bring barriers tolanguage learning and reduce learners learning capacity. Withtwo true stories as an introduction and some relevant answersobtained from my questionnaire, this paper mainly discusses theinfluences of negative emotional factors on language learning.such as anxiety, low self-esteem, insecure classroomatmosphere, lack of rapport between teachers and students, etc.Some suggestions about how to overcome affective barriers areput forward.

  6. Does Political Ideology Affect Economic Growth?

    DEFF Research Database (Denmark)

    Bjørnskov, Christian

    2005-01-01

    their political ideology, affect economic performance. The paper presents evidence suggesting that rightwing societies have grown faster in the last decades than other democratic societies. Further analysis suggests that these societies develop better legal systems and less government intervention, which in turn......This paper asks the question whether political ideology affects economic growth. Voters may demand inefficient levels of redistribution and government intervention, and they may care too little for aspects that really matter for the economy. Their norms and perceptions of society might, via...

  7. Affective Variables of Second Language Learning

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    <正>There are various factors influencing second language learning(L2 learning). Among then,affective variables directly or indirectly have an effect on the success of L2 learning. Based on the applied linguistics theories,the paper mainly discusses three major groups of affective variables—attitude,motivation,personality and how they influence L2 learning.It aims to make both language teachers and learners understand the importance of these variables so as to improve the efficiency of L2 learning and teaching.

  8. Does temperament affect learning in calves?

    DEFF Research Database (Denmark)

    Webb, Laura E.; van Reenen, Cornelis G.; Jensen, Margit Bak;

    2015-01-01

    challenge tests, may affect learning an operant conditioning task in calves. Understanding how temperament affects learning in calves can help with the training of calves on novel automated feeding apparatuses or on novel feed components, and can thus help improve calf health and welfare....... on learning variables, and these were proposed to reflect feed motivation and working speed. A single correlation was found between temperament and learning PC scores: high activity was associated with low feed motivation. This preliminary exploratory study suggests that temperament, as assessed during...

  9. Mood swings: design and evaluation of affective interactive art

    NARCIS (Netherlands)

    Bialoskorski, Leticia S.S.; Westerink, Joyce H.D.M.; Broek, van den Egon L.

    2009-01-01

    The field of affective computing is concerned with developing emphatic products, such as affective consumer products, affective games, and affective art. This paper describes Mood Swings, an affective interactive art system, which interprets and visualizes affect expressed by a person. Mood Swings c

  10. Positive affect, negative affect, stress, and social support as mediators of the forgiveness-health relationship.

    Science.gov (United States)

    Green, Michelle; Decourville, Nancy; Sadava, Stanley

    2012-01-01

    Structural equation modeling was used to test a model in which positive affect, negative affect, perceived stress, and social support were hypothesized to mediate the relationship between forgiveness and mental and physical health. Six hundred and twenty-three undergraduates completed a battery of self-report measures. Results of the analyses indicated that the forgiveness-health relation was mediated by positive affect, negative affect, stress, and the interrelationship between negative affect and stress. There was limited support for social support and the interrelationship between positive affect and social support as mediators. The results suggested that the relationship between forgiveness and health is mediated rather than direct. Implications and directions for future research are discussed.

  11. Role of affective attitudes and anticipated affective reactions in predicting health behaviors

    OpenAIRE

    Conner, M; McEachan, R; Taylor, N.; O'Hara, J; Lawton, R

    2015-01-01

    Objective: Two measures of affect-affective attitude (AA) and anticipated affective reaction (AAR)- have frequently been used individually, but rarely simultaneously, in correlational studies predicting health behaviors. This research assessed their individual and combined impact in predicting intention and action for a range of health behaviors, controlling for theory of planned behavior (TPB) variables. Method: Self-reported intentions and performance of health behaviors were the main outco...

  12. Mechanisms of DNA repair and radio-induced mutagenesis in higher eukaryotes; Mecanismes de reparation et mutagenese radio-induite chez les eucaryotes superieurs

    Energy Technology Data Exchange (ETDEWEB)

    Averbeck, D. [Centre Universitaire d' Orsay, Institut Curie, Section de Recherche, Lab. Raymond-Latarjet, UMR 2027 CNRS, 91 (France)

    2000-10-01

    Cells of higher eukaryotes possess several very efficient systems for the repair of radiation-induced lesions in DNA. Different strategies have been adopted at the cellular level to remove or even tolerate various types of lesions in order to assure survival and limit the mutagenic consequences. In mammalian cells, the main DNA repair systems comprise direct reversion of damage, excision of damage and exchange mechanisms with intact DNA. Among these, the direct ligation of single strand breaks (SSB) by a DNA ligase and the multi-enzymatic repair systems of mismatch repair, base and nucleotide excision repair as well as the repair of double strand breaks (DSB) by homologous recombination or non homologous end-joining are the most important systems. Most of these processes are error-free except the non homologous end-joining pathway used for the repair of DSB. Moreover, certain lesions can be tolerated by more or less accurately acting polymerases capable of performing trans-lesion DNA syntheses. The DNA repair systems are intimately integrated in the network of cellular regulation. Some of their components are DNA damage inducible. Radiation-induced mutagenesis is largely due to unrepaired DNA damage but also involves error-prone repair processes like the repair of DSB by non-homologous end-joining. Generally, mammalian cells are well prepared to repair radiation-induced lesions. However, some questions remain to be asked about mechanistic details and efficiencies of the systems for removing certain types of radiation-damage and about their order and timing of action. The answers to these questions would be important for radioprotection as well as radiotherapy. (author)

  13. Questions of time and affect: a person's affectivity profile, time perspective, and well-being.

    Science.gov (United States)

    Garcia, Danilo; Sailer, Uta; Nima, Ali Al; Archer, Trevor

    2016-01-01

    Background. A "balanced" time perspective has been suggested to have a positive influence on well-being: a sentimental and positive view of the past (high Past Positive), a less pessimistic attitude toward the past (low Past Negative), the desire of experiencing pleasure with slight concern for future consequences (high Present Hedonistic), a less fatalistic and hopeless view of the future (low Present Fatalistic), and the ability to find reward in achieving specific long-term goals (high Future). We used the affective profiles model (i.e., combinations of individuals' experience of high/low positive/negative affectivity) to investigate differences between individuals in time perspective dimensions and to investigate if the influence of time perspective dimensions on well-being was moderated by the individual's type of profile. Method. Participants (N = 720) answered to the Positive Affect Negative Affect Schedule, the Zimbardo Time Perspective Inventory and two measures of well-being: the Temporal Satisfaction with Life Scale and Ryff's Scales of Psychological Well-Being-short version. A Multivariate Analysis of Variance (MANOVA) was conducted to identify differences in time perspective dimensions and well-being among individuals with distinct affective profiles. Four structural equation models (SEM) were used to investigate which time perspective dimensions predicted well-being for individuals in each profile. Results. Comparisons between individuals at the extreme of the affective profiles model suggested that individuals with a self-fulfilling profile (high positive/low negative affect) were characterized by a "balanced" time perspective and higher well-being compared to individuals with a self-destructive profile (low positive/high negative affect). However, a different pattern emerged when individuals who differed in one affect dimension but matched in the other were compared to each other. For instance, decreases in the past negative time perspective

  14. Assessing affective variability in eating disorders: affect spins less in anorexia nervosa of the restrictive type.

    Science.gov (United States)

    Vansteelandt, Kristof; Probst, Michel; Pieters, Guido

    2013-08-01

    Differences in affective variability in eating disorders are examined using an ecological momentary assessment (EMA) protocol. It is hypothesized that restriction serves to pre-empt the activation of affect whereas bulimic behavior serves to cope with overwhelming affect once activated. Therefore, we expect anorexia nervosa (AN) patients of the restricting type (AN-RT) to have lower mean levels of affect and less affective variability than Bulimia Nervosa (BN) patients. Patients' successive affective states over time are represented as different positions in a two-dimensional space defined by the orthogonal dimensions of valence and activation. Affective variability is measured by the within person variance and the new concepts of pulse and spin. Results of this exploratory study suggest that the diagnostic groups have the same mean levels of affect but affect spins less in patients with AN-RT. Using an EMA protocol and measures like pulse and spin may reveal insights in eating disorders that remain hidden with more traditional assessment methods.

  15. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system.

    Science.gov (United States)

    Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

    2013-10-11

    Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

  16. TALE nucleases and next generation GM crops.

    KAUST Repository

    Mahfouz, Magdy M.

    2011-04-01

    Site-specific and adaptable DNA binding domains are essential modules to develop genome engineering technologies for crop improvement. Transcription activator-like effectors (TALEs) proteins are used to provide a highly specific and adaptable DNA binding modules. TALE chimeric nucleases (TALENs) were used to generate site-specific double strand breaks (DSBs) in vitro and in yeast, Caenorhabditis elegans, mammalian and plant cells. The genomic DSBs can be generated at predefined and user-selected loci and repaired by either the non-homologous end joining (NHEJ) or homology dependent repair (HDR). Thus, TALENs can be used to achieve site-specific gene addition, stacking, deletion or inactivation. TALE-based genome engineering tools should be powerful to develop new agricultural biotechnology approaches for crop improvement. Here, we discuss the recent research and the potential applications of TALENs to accelerate the generation of genomic variants through targeted mutagenesis and to produce a non-transgenic GM crops with the desired phenotype.

  17. Stem Cells: The Pursuit of Genomic Stability

    Directory of Open Access Journals (Sweden)

    Saranya P. Wyles

    2014-11-01

    Full Text Available Stem cells harbor significant potential for regenerative medicine as well as basic and clinical translational research. Prior to harnessing their reparative nature for degenerative diseases, concerns regarding their genetic integrity and mutation acquisition need to be addressed. Here we review pluripotent and multipotent stem cell response to DNA damage including differences in DNA repair kinetics, specific repair pathways (homologous recombination vs. non-homologous end joining, and apoptotic sensitivity. We also describe DNA damage and repair strategies during reprogramming and discuss potential genotoxic agents that can reduce the inherent risk for teratoma formation and mutation accumulation. Ensuring genomic stability in stem cell lines is required to achieve the quality control standards for safe clinical application.

  18. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

    Science.gov (United States)

    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

  19. Control of gene editing by manipulation of DNA repair mechanisms.

    Science.gov (United States)

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  20. DNA repair mechanisms in eukaryotes: Special focus in Entamoeba histolytica and related protozoan parasites.

    Science.gov (United States)

    López-Camarillo, César; Lopez-Casamichana, Mavil; Weber, Christian; Guillen, Nancy; Orozco, Esther; Marchat, Laurence A

    2009-12-01

    Eukaryotic cell viability highly relies on genome stability and DNA integrity maintenance. The cellular response to DNA damage mainly consists of six biological conserved pathways known as homologous recombination repair (HRR), non-homologous end-joining (NHEJ), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and methyltransferase repair that operate in a concerted way to minimize genetic information loss due to a DNA lesion. Particularly, protozoan parasites survival depends on DNA repair mechanisms that constantly supervise chromosomes to correct damaged nucleotides generated by cytotoxic agents, host immune pressure or cellular processes. Here we reviewed the current knowledge about DNA repair mechanisms in the most relevant human protozoan pathogens. Additionally, we described the recent advances to understand DNA repair mechanisms in Entamoeba histolytica with special emphasis in the use of genomic approaches based on bioinformatic analysis of parasite genome sequence and microarrays technology.

  1. A molecular genetic toolbox for Yarrowia lipolytica

    DEFF Research Database (Denmark)

    Bredeweg, Erin L.; Pomraning, Kyle R.; Dai, Ziyu

    2017-01-01

    used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment...... of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products........ Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted...

  2. CRISPR-Cas9-Guided Genome Engineering in C. elegans.

    Science.gov (United States)

    Kim, Hyun-Min; Colaiácovo, Monica P

    2016-07-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans. Recent studies have developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc.

  3. The DNA repair complex DNA-PK, a pharmacological target in cancer chemotherapy and radiotherapy; Le complexe de reparation de l'ADN DNA-PK, une cible pharmacologique en chimiotherapie et radiotherapie anticancereuse

    Energy Technology Data Exchange (ETDEWEB)

    Salles, B.; Calsou, P.; Frit, P.; Muller, C. [Institut de Pharmacologie et Biologie Structurale (IPBS), UMR CNRS 5089, 31 - Toulouse (France)

    2006-05-15

    A line of investigation in the search for sensitizing tumor cells to chemotherapy or radiotherapy relies on the selection of DNA repair inhibitors. In the area of DNA repair mechanisms, DNA-dependent protein kinase (DNA-PK) represents a key complex. Indeed DNA-PK is involved in the non-homologous end joining (NHEJ) process that corresponds to the major activity responsible for cell survival after ionizing radiation or chemotherapeutic treatment producing DNA double strand breaks. DNA-PK belongs to the PI3-K related kinase family and specific inhibitors have been recently selected and evaluated as radio- and chemo-sensitizers. These drugs, along with other ways to inhibit the DSBs repair process, are presented and discussed. (authors)

  4. In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

    DEFF Research Database (Denmark)

    Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens;

    2008-01-01

    BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins...... in the nucleus with no detectable amount in the cytoplasm. This distribution was not altered upon irradiation. In non-irradiated cells the level of these proteins varied with a CV ranging between 16% and 20%, but there was no correlation with the respective cellular DSB repair capacity. Irradiation (3.5 and 15Gy......) did not alter the expression of these proteins and there was also no change in the DNA-PK activity. These results indicate that the variation in DSB repair capacity determined for these fibroblasts can be ascribed to differences neither in the localisation or expression of Ku70, Ku80 and XRCC4 nor...

  5. SCAI promotes DNA double-strand break repair in distinct chromosomal contexts

    DEFF Research Database (Denmark)

    Hansen, Rebecca Kring; Mund, Andreas; Poulsen, Sara Lund;

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer...... cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1......-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs...

  6. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

    Directory of Open Access Journals (Sweden)

    Zita Nagy

    2016-02-01

    Full Text Available 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.

  7. 53BP1 fosters fidelity of homology-directed DNA repair

    DEFF Research Database (Denmark)

    Ochs, Fena; Somyajit, Kumar; Altmeyer, Matthias;

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair...... (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1...... fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome...

  8. 53BP1 regulates DSB repair using Rif1 to control 5' end resection.

    Science.gov (United States)

    Zimmermann, Michal; Lottersberger, Francisca; Buonomo, Sara B; Sfeir, Agnel; de Lange, Titia

    2013-02-08

    The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or nonhomologous end joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ, and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1; limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage; and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.

  9. [DNA homologous recombination repair in mammalian cells].

    Science.gov (United States)

    Popławski, Tomasz; Błasiak, Janusz

    2006-01-01

    DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.

  10. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    K. C. Summers

    2011-01-01

    Full Text Available Repair of double-stranded breaks (DSBs is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ, homologous recombination (HR, or the inclusive DNA damage response (DDR. These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

  11. [The application of genome editing in identification of plant gene function and crop breeding].

    Science.gov (United States)

    Xiangchun, Zhou; Yongzhong, Xing

    2016-03-01

    Plant genome can be modified via current biotechnology with high specificity and excellent efficiency. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system are the key engineered nucleases used in the genome editing. Genome editing techniques enable gene targeted mutagenesis, gene knock-out, gene insertion or replacement at the target sites during the endogenous DNA repair process, including non-homologous end joining (NHEJ) and homologous recombination (HR), triggered by the induction of DNA double-strand break (DSB). Genome editing has been successfully applied in the genome modification of diverse plant species, such as Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. In this review, we summarize the application of genome editing in identification of plant gene function and crop breeding. Moreover, we also discuss the improving points of genome editing in crop precision genetic improvement for further study.

  12. [Application of CRISPR-Cas9 genome editing for constructing animal models of human diseases].

    Science.gov (United States)

    Ou, Zhanhui; Sun, Xiaofang

    2016-08-01

    The CRISPR-Cas9 system is a new targeted nuclease for genome editing, which can directly introduce modifications at the targeted genomic locus. The system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 in the genome. Upon targeting, Cas9 can generate DNA double-strand breaks (DSBs). As such DSBs are repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR), therefore facilitates introduction of random or specific mutations, repair of endogenous mutations, or insertion of DNA elements. The system has been successfully used to generate gene targeted cell lines including those of human, animals and plants. This article reviews recent advances made in this rapidly evolving technique for the generation of animal models for human diseases.

  13. DNA End Resection:Facts and Mechanisms

    Institute of Scientific and Technical Information of China (English)

    Ting Liu; a Jun Huang; b

    2016-01-01

    DNA double-strand breaks (DSBs), which arise following exposure to a number of endogenous and exogenous agents, can be repaired by either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways in eukaryotic cells. A vital step in HR repair is DNA end resection, which generates a long 30 single-stranded DNA (ssDNA) tail that can invade the homologous DNA strand. The generation of 30 ssDNA is not only essential for HR repair, but also promotes activation of the ataxia telangiectasia and Rad3-related protein (ATR). Multiple fac-tors, including the MRN/X complex, C-terminal-binding protein interacting protein (CtIP)/Sae2, exonuclease 1 (EXO1), Bloom syndrome protein (BLM)/Sgs1, DNA2 nuclease/helicase, and several chromatin remodelers, cooperate to complete the process of end resection. Here we review the basic machinery involved in DNA end resection in eukaryotic cells.

  14. Genome engineering with targetable nucleases.

    Science.gov (United States)

    Carroll, Dana

    2014-01-01

    Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for their use in different biological systems.

  15. Mutations in XRCC4 cause primordial dwarfism without causing immunodeficiency.

    Science.gov (United States)

    Saito, Shinta; Kurosawa, Aya; Adachi, Noritaka

    2016-08-01

    In successive reports from 2014 to 2015, X-ray repair cross-complementing protein 4 (XRCC4) has been identified as a novel causative gene of primordial dwarfism. XRCC4 is indispensable for non-homologous end joining (NHEJ), the major pathway for repairing DNA double-strand breaks. As NHEJ is essential for V(D)J recombination during lymphocyte development, it is generally believed that abnormalities in XRCC4 cause severe combined immunodeficiency. Contrary to expectations, however, no overt immunodeficiency has been observed in patients with primordial dwarfism harboring XRCC4 mutations. Here, we describe the various XRCC4 mutations that lead to disease and discuss their impact on NHEJ and V(D)J recombination.

  16. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

    Science.gov (United States)

    Aymard, François; Bugler, Beatrix; Schmidt, Christine K; Guillou, Emmanuelle; Caron, Pierre; Briois, Sébastien; Iacovoni, Jason S; Daburon, Virginie; Miller, Kyle M; Jackson, Stephen P; Legube, Gaëlle

    2014-04-01

    Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

  17. ATM Dependent Silencing Links Nucleolar Chromatin Reorganization to DNA Damage Recognition.

    Science.gov (United States)

    Harding, Shane M; Boiarsky, Jonathan A; Greenberg, Roger A

    2015-10-13

    Resolution of DNA double-strand breaks (DSBs) is essential for the suppression of genome instability. DSB repair in transcriptionally active genomic regions represents a unique challenge that is associated with ataxia telangiectasia mutated (ATM) kinase-mediated transcriptional silencing. Despite emerging insights into the underlying mechanisms, how DSB silencing connects to DNA repair remains undefined. We observe that silencing within the rDNA depends on persistent DSBs. Non-homologous end-joining was the predominant mode of DSB repair allowing transcription to resume. ATM-dependent rDNA silencing in the presence of persistent DSBs led to the large-scale reorganization of nucleolar architecture, with movement of damaged chromatin to nucleolar cap regions. These findings identify ATM-dependent temporal and spatial control of DNA repair and provide insights into how communication between DSB signaling and ongoing transcription promotes genome integrity.

  18. ATM Dependent Silencing Links Nucleolar Chromatin Reorganization to DNA Damage Recognition

    Directory of Open Access Journals (Sweden)

    Shane M. Harding

    2015-10-01

    Full Text Available Resolution of DNA double-strand breaks (DSBs is essential for the suppression of genome instability. DSB repair in transcriptionally active genomic regions represents a unique challenge that is associated with ataxia telangiectasia mutated (ATM kinase-mediated transcriptional silencing. Despite emerging insights into the underlying mechanisms, how DSB silencing connects to DNA repair remains undefined. We observe that silencing within the rDNA depends on persistent DSBs. Non-homologous end-joining was the predominant mode of DSB repair allowing transcription to resume. ATM-dependent rDNA silencing in the presence of persistent DSBs led to the large-scale reorganization of nucleolar architecture, with movement of damaged chromatin to nucleolar cap regions. These findings identify ATM-dependent temporal and spatial control of DNA repair and provide insights into how communication between DSB signaling and ongoing transcription promotes genome integrity.

  19. Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation

    DEFF Research Database (Denmark)

    Ghamrasni, S El; Cardoso, R; Li, L;

    2016-01-01

    Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA......-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent....... The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights...

  20. TALE nucleases and next generation GM crops.

    Science.gov (United States)

    Mahfouz, Magdy M; Li, Lixin

    2011-01-01

    Site-specific and adaptable DNA binding domains are essential modules to develop genome engineering technologies for crop improvement. Transcription activator-like effectors (TALEs) proteins are used to provide a highly specific and adaptable DNA binding modules. TALE chimeric nucleases (TALENs) were used to generate site-specific double strand breaks (DSBs) in vitro and in yeast, Caenorhabditis elegans, mammalian and plant cells. The genomic DSBs can be generated at predefined and user-selected loci and repaired by either the non-homologous end joining (NHEJ) or homology dependent repair (HDR). Thus, TALENs can be used to achieve site-specific gene addition, stacking, deletion or inactivation. TALE-based genome engineering tools should be powerful to develop new agricultural biotechnology approaches for crop improvement. Here, we discuss the recent research and the potential applications of TALENs to accelerate the generation of genomic variants through targeted mutagenesis and to produce a non-transgenic GM crops with the desired phenotype.

  1. DNA double-strand break repair: a tale of pathway choices

    Institute of Scientific and Technical Information of China (English)

    Jing Li; Xingzhi Xu

    2016-01-01

    Deoxyribonucleic acid double-strand breaks (DSBs) are cytotoxic lesions that must be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways.DSB repair is critical for genome integrity,cellular homeostasis and also constitutes the biological foundation for radiotherapy and the majority of chemotherapy.The choice between HR and NHEJ is a complex yet not completely understood process that will entail more future efforts.Herein we review our current understandings about how the choice is made over an antagonizing balance between p53-binding protein 1 and breast cancer 1 in the context of cell cycle stages,downstream effects,and distinct chromosomal histone marks.These exciting areas of research will surely bring more mechanistic insights about DSB repair and be utilized in the clinical settings.

  2. DNA ligase IV as a new molecular target for temozolomide

    Energy Technology Data Exchange (ETDEWEB)

    Kondo, Natsuko [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Department of Neurosurgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa; Mori, Eiichiro; Ohnishi, Ken [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); McKinnon, Peter J. [Department of Genetics and Tumor Cell Biology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Sakaki, Toshisuke; Nakase, Hiroyuki [Department of Neurosurgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Takeo, E-mail: tohnishi@naramed-u.ac.jp [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)

    2009-10-02

    Temozolomide (TMZ) is a methylating agent used in chemotherapy against glioblastoma. This work was designed to clarify details in repair pathways acting to remove DNA double-strand breaks (DSBs) induced by TMZ. Cultured mouse embryonic fibroblasts were used which were deficient in DSB repair genes such as homologous recombination repair-related genes X-ray repair cross-complementing group 2 (XRCC2)and radiation sensitive mutant54 (Rad54), non-homologous end joining repair-related gene DNAligase IV (Lig4). Cell sensitivity to drug treatments was assessed using colony forming assays. The most effective molecular target which was correlated with TMZ cell sensitivity was Lig4. In addition, it was found that small interference RNAs (siRNA) for Lig4 efficiently enhanced cell lethality induced by TMZ in human glioblastoma A172 cells. These findings suggest that down regulation of Lig4 might provide a useful tool for cell sensitization during TMZ chemotherapy.

  3. Structure of Bacterial LigD -phosphoesterase Unveils a DNA Repair Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Nair, P.; Smith, P; Shuman, S

    2010-01-01

    The DNA ligase D (LigD) 3{prime}-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3{prime} end-healing reactions at DNA double-strand breaks. Here we report the 1.9 {angstrom} crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded {beta} barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis a vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.

  4. Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System.

    Directory of Open Access Journals (Sweden)

    Gary Alan Barclay Armstrong

    Full Text Available The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated protein 9 type II (Cas9 system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.

  5. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  6. Challenges to increasing targeting efficiency in genome engineering.

    Science.gov (United States)

    Horii, Takuro; Hatada, Izuho

    2016-01-01

    Gene targeting technologies are essential for the analysis of gene functions. Knockout mouse generation via genetic modification of embryonic stem cells (ESCs) is the commonest example, but it is a time-consuming and labor-intensive procedure. Recently, a novel genome editing technology called CRISPR/Cas has enabled the direct production of knockout mice by non-homologous end joining (NHEJ)-mediated mutations. Unexpectedly, however, it generally exhibits a low efficiency in homologous recombination (HR) and is prone to high mosaicism. Meanwhile, gene targeting using ESCs is still being improved, as reported by Fukuda et al. in this issue. Here, we outline current gene targeting technologies with special emphasis on HR-mediated technologies, which are currently being performed using these two major strategies.

  7. SunnyTALEN: a second-generation TALEN system for human genome editing.

    Science.gov (United States)

    Sun, Ning; Bao, Zehua; Xiong, Xiong; Zhao, Huimin

    2014-04-01

    Transcription activator-like effector nucleases (TALENs) have rapidly emerged as a powerful genome editing tool. The site-specific DNA double-strand breaks generated by TALENs in the human chromosome can induce homologous recombination or non-homologous end joining, resulting in desired genetic modifications. In this study, we report the development of a TALEN variant, SunnyTALEN, with >2.5-fold improved genome editing efficacy in human cells. The corresponding scaffold increases the rate of genetic modification at all the 13 tested loci of human genome and is compatible with heterodimer TALEN architectures. This enhanced and high-efficiency TALEN variant represents a novel second-generation TALEN system and has great potential for biological and therapeutic applications.

  8. TALEN utilization in rice genome modifications.

    Science.gov (United States)

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2014-08-15

    Transcription activator-like effector nucleases (TALENs), the newly developed and powerful genetic tools for precise genome editing, are fusion proteins of TAL effectors as DNA binding domains and the cleavage domain of FokI endonuclease. As a pair, the central repeat regions of TALENs determine the DNA binding specificity for the two sub-target sites; and the dimeric non-specific FokI cleavage domains cause a DNA double strand break (DSB) between the bound sequences. In vivo, cells repair the DSBs through either non-homologous end joining (NHEJ) pathway or homologous recombination (HR) pathway. Various methods have been developed for easy and fast assembly of TALEN genes for their utilization in a variety of eukaryotic cells or organisms. Here we present a TALEN-based rice genome modification protocol including constructing modularly assembled TALENs, rice transformation, and mutant screening.

  9. Synthetic lethality between PAXX and XLF in mammalian development

    Science.gov (United States)

    Balmus, Gabriel; Barros, Ana C.; Wijnhoven, Paul W.G.; Lescale, Chloé; Hasse, Hélène Lenden; Boroviak, Katharina; le Sage, Carlos; Doe, Brendan; Speak, Anneliese O.; Galli, Antonella; Jacobsen, Matt; Deriano, Ludovic; Adams, David J.; Jackson, Stephen P.

    2016-01-01

    PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf−/− mice, Paxx−/− mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4−/− and Lig4−/− mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals. PMID:27798842

  10. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Science.gov (United States)

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  11. RNF4 is required for DNA double-strand break repair in vivo

    DEFF Research Database (Denmark)

    Vyas, R; Kumar, R; Clermont, F

    2013-01-01

    for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling......Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial......, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations...

  12. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2011-01-01

    : homologous recombination and non-homologous end-joining. Ataxia telangiectasia and related disorders with defects in these pathways illustrate that such defects can lead to early childhood neurodegeneration. Aging is a risk factor for neurodegeneration and accumulation of oxidative mitochondrial DNA damage......Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive...... neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative...

  13. APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Manikandan Periyasamy

    2015-10-01

    Full Text Available Estrogen receptor α (ERα is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER and to repair by non-homologous end-joining (NHEJ pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

  14. How does concurrent sourcing affect performance?

    DEFF Research Database (Denmark)

    Mols, Niels Peter

    2010-01-01

    Abstract Purpose – “Concurrent sourcing” is a term used by Parmigiani to describe the phenomenon where a firm simultaneously buys and makes the same good or service. The purpose of this paper is to develop propositions that suggest how concurrent sourcing affects performance. Design/methodology/a...

  15. Water hardness affects catfish susceptibility to columnaris

    Science.gov (United States)

    Columnaris disease can cause tremendous losses of freshwater fish. While it has been studied exhaustively, little is known about its affinity to specific water chemistries that affects attachment. Recent studies in our labs have illuminated this subject. In the first experiment, two waters were ...

  16. Healthcare resource allocation decisions affecting uninsured services

    Science.gov (United States)

    Harrison, Krista Lyn; Taylor, Holly A.

    2017-01-01

    Purpose Using the example of community access programs (CAPs), the purpose of this paper is to describe resource allocation and policy decisions related to providing health services for the uninsured in the USA and the organizational values affecting these decisions. Design/methodology/approach The study used comparative case study methodology at two geographically diverse sites. Researchers collected data from program documents, meeting observations, and interviews with program stakeholders. Findings Five resource allocation or policy decisions relevant to providing healthcare services were described at each site across three categories: designing the health plan, reacting to funding changes, and revising policies. Organizational values of access to care and stewardship most frequently affected resource allocation and policy decisions, while economic and political pressures affect the relative prioritization of values. Research limitations/implications Small sample size, the potential for social desirability or recall bias, and the exclusion of provider, member or community perspectives beyond those represented among participating board members. Practical implications Program directors or researchers can use this study to assess the extent to which resource allocation and policy decisions align with organizational values and mission statements. Social implications The description of how healthcare decisions are actually made can be matched with literature that describes how healthcare resource decisions ought to be made, in order to provide a normative grounding for future decisions. Originality/value This study addresses a gap in literature regarding how CAPs actually make resource allocation decisions that affect access to healthcare services. PMID:27934550

  17. Fight or flight: Affective news framing effects

    NARCIS (Netherlands)

    Feinholdt, A.

    2016-01-01

    This dissertation investigates how emotions shape and are shaped by news framing and how this interplay leads to a number of affective news framing effects. My work reveals that emotions do not only function as mechanisms but also as channels strengthening or weakening framing effects. In addition,

  18. Gamma flicker elicits positive affect without awareness

    NARCIS (Netherlands)

    B.T. Heerebout; A.E.Y Tap; M. Rotteveel; R.H. Phaf

    2013-01-01

    High-frequency oscillations emerged as a neural code for both positive affect and fluent attentional processing from evolutionary simulations with artificial neural networks. Visual 50 Hz flicker, which entrains neural oscillations in the gamma band, has been shown to foster attentional switching, b

  19. Environmental Factors Affecting Preschoolers' Motor Development

    Science.gov (United States)

    Venetsanou, Fotini; Kambas, Antonis

    2010-01-01

    The process of development occurs according to the pattern established by the genetic potential and also by the influence of environmental factors. The aim of the present study was to focus on the main environmental factors affecting motor development. The review of the literature revealed that family features, such as socioeconomic status,…

  20. Applied Stress Affecting the Environmentally Assisted Cracking

    Science.gov (United States)

    Vasudevan, A. K.

    2013-03-01

    Stress corrosion cracking (SCC) is affected by the mode of applied stress, i.e., tension, compression, or torsion. The cracking is measured in terms of initiation time to nucleate a crack or time to failure. In a simple uniaxial loading under tension or compression, it is observed that the initiation time can vary in orders of magnitude depending on the alloy and the environment. Fracture can be intergranular or transgranular or mixed mode. Factors that affect SCC are solubility of the metal into surrounding chemical solution, and diffusion rate (like hydrogen into a tensile region) of an aggressive element into the metal and liquid metallic elements in the grain boundaries. Strain hardening exponent that affects the local internal stresses and their gradients can affect the diffusion kinetics. We examine two environments (Ga and 3.5 pct NaCl) for the same alloy 7075-T651, under constant uniaxial tension and compression load. These two cases provide us application to two different governing mechanisms namely liquid metal embrittlement (7075-Ga) and hydrogen-assisted cracking (7075-NaCl). We note that, in spite of the differences in their mechanisms, both systems show similar behavior in the applied K vs crack initiation time plots. One common theme among them is the transport mechanism of a solute element to a tensile-stress region to initiate fracture.

  1. Affective Education for Visually Impaired Children.

    Science.gov (United States)

    Locke, Don C.; Gerler, Edwin R., Jr.

    1981-01-01

    Evaluated the effectiveness of the Human Development Program (HDP) and the Developing Understanding of Self and Others (DUSO) program used with visually impaired children. Although HDP and DUSO affected the behavior of visually impaired children, they did not have any effect on children's attitudes toward school. (RC)

  2. The Affective Experience of Novice Computer Programmers

    Science.gov (United States)

    Bosch, Nigel; D'Mello, Sidney

    2017-01-01

    Novice students (N = 99) participated in a lab study in which they learned the fundamentals of computer programming in Python using a self-paced computerized learning environment involving a 25-min scaffolded learning phase and a 10-min unscaffolded fadeout phase. Students provided affect judgments at approximately 100 points (every 15 s) over the…

  3. Physical conditions affecting pyrethroid toxicity in arthropods.

    NARCIS (Netherlands)

    Jagers op Akkerhuis, G.A.J.M.

    1993-01-01

    The aim of this thesis was to obtain mechanistic information about how the toxicity of pesticides in the field is affected by physical factors, pesticide bioavailability and arthropod behaviour. The pyrethroid insecticide deltamethrin and linyphiid spiders were selected as pesticide-effect model. In

  4. How gluten properties are affected by pentosans

    NARCIS (Netherlands)

    Wang, M.; Vliet, T. van; Hamer, R.J.

    2004-01-01

    During the wet separation of starch and gluten, both water extractable pentosans (WEP) and water unextractable solids (WUS) have a negative effect on gluten yield. Gluten properties are also affected: the gluten becomes less extensible. In comparison to the control, addition of WUS or WEP resulted i

  5. Infrasound from Wind Turbines Could Affect Humans

    Science.gov (United States)

    Salt, Alec N.; Kaltenbach, James A.

    2011-01-01

    Wind turbines generate low-frequency sounds that affect the ear. The ear is superficially similar to a microphone, converting mechanical sound waves into electrical signals, but does this by complex physiologic processes. Serious misconceptions about low-frequency sound and the ear have resulted from a failure to consider in detail how the ear…

  6. Human Sexuality: An Affective Classroom Approach

    Science.gov (United States)

    Ackerman, Kathi; Gumaer, Jim

    1976-01-01

    The affective education program on human sexuality in this study increased students' awareness about the human body without increasing feelings of anxiety. The sessions provided an atmosphere which helped students understand themselves better and increase their knowledge about personal interests. (Author)

  7. Generating Reliable and Affective Choreography through Engineering

    DEFF Research Database (Denmark)

    Jochum, Elizabeth

    How do we define graceful motion? Is grace exclusively the province of living, sentient beings, or is it possible to automate graceful motion? The GRACE project (Generating Reliable and Affective Choreography through Engineering) uses these two questions to investigate what makes movement graceful...

  8. [Affective disorders: endocrine and metabolic comorbidities].

    Science.gov (United States)

    Cermolacce, M; Belzeaux, R; Adida, M; Azorin, J-M

    2014-12-01

    Links between affective and endocrine-metabolic disorders are numerous and complex. In this review, we explore most frequent endocrine-metabolic comorbidities. On the one hand, these comorbidities imply numerous iatrogenic effects from antipsychotics (metabolic side-effects) or from lithium (endocrine side-effects). On the other hand, these comorbidities are also associated with affective disorders independently from medication. We will successively examine metabolic syndrome, glycemic disturbances, obesity and thyroid disorders among patients with affective disorders. Endocrinemetabolic comorbidities can be individually encountered, but can also be associated. Therefore, they substantially impact morbidity and mortality by increasing cardiovascular risk factors. Two distinct approaches give an account of processes involved in these comorbidities: common environmental factors (iatrogenic effects, lifestyle), and/or shared physiological vulnerabilities. In conclusion, we provide a synthesis of important results and recommendations related to endocrine-metabolic comorbidities in affective disorders : heavy influence on morbidity and mortality, undertreatment of somatic diseases, importance of endocrine and metabolic side effects from main mood stabilizers, impact from sex and age on the prevalence of comorbidities, influence from previous depressive episodes in bipolar disorders, and relevance of systematic screening for subclinical (biological) disturbances.

  9. Environmental issues affecting clean coal technology deployment

    Energy Technology Data Exchange (ETDEWEB)

    Miller, M.J. [Electric Power Research Inst., Palo Alto, CA (United States)

    1997-12-31

    The author outlines what he considers to be the key environmental issues affecting Clean Coal Technology (CCT) deployment both in the US and internationally. Since the international issues are difficult to characterize given different environmental drivers in various countries and regions, the primary focus of his remarks is on US deployment. However, he makes some general remarks, particularly regarding the environmental issues in developing vs. developed countries and how these issues may affect CCT deployment. Further, how environment affects deployment depends on which particular type of clean coal technology one is addressing. It is not the author`s intention to mention many specific technologies other than to use them for the purposes of example. He generally categorizes CCTs into four groups since environment is likely to affect deployment for each category somewhat differently. These four categories are: Precombustion technologies such as coal cleaning; Combustion technologies such as low NOx burners; Postcombustion technologies such as FGD systems and postcombustion NOx control; and New generation technologies such as gasification and fluidized bed combustion.

  10. 47 CFR 1.2003 - Applications affected.

    Science.gov (United States)

    2010-10-01

    ... Abuse Act of 1988 § 1.2003 Applications affected. The certification required by § 1.2002 must be filed... and/or Response Station(s) and Low Power Relay Station(s) License; FCC 340Application for Construction... a Low Power Television Station; FCC 346Application for Authority to Construct or Make Changes in...

  11. [How does summer affect sexual desire?].

    Science.gov (United States)

    Kontula, Osmo; Väisälä, Leena

    2013-01-01

    Sexual desire involves many different things such as sexual thoughts and images, excitement, expectation and orgasm. Mood has a strong association with sexual desire. Fatigue and depression in particular cause lack of sexual desire. By affecting the state of alertness and energy in humans, sunlight may increase sexual activity.

  12. Go Naked: Diapers Affect Infant Walking

    Science.gov (United States)

    Cole, Whitney G.; Lingeman, Jesse M.; Adolph, Karen E.

    2012-01-01

    In light of cross-cultural and experimental research highlighting effects of childrearing practices on infant motor skill, we asked whether wearing diapers, a seemingly innocuous childrearing practice, affects infant walking. Diapers introduce bulk between the legs, potentially exacerbating infants' poor balance and wide stance. We show that…

  13. Attention to Affect in Language Learning

    Science.gov (United States)

    Arnold, Jane

    2011-01-01

    As language teachers, we have to pay attention to many things in our work so why add "attention to affect"? Perhaps the simplest, most direct answer is that whatever we focus most on in our particular context, be it general English, morphosyntax, phonetics, literature, English for academic writing or any other special area, attention to…

  14. How Going to School Affects the Family

    DEFF Research Database (Denmark)

    Landersø, Rasmus; Nielsen, Helena Skyt; Simonsen, Marianne

    in school starting age induced by date of birth, we find that the timing of transitions affect parental outcomes - including marriage and maternal employment - and older siblings’ academic performance. Our results indicate that families redistribute resources across the entire family in response to a single...

  15. Trauma and suicidality in war affected communities

    NARCIS (Netherlands)

    J. Jankovic; S. Bremner; M. Bogic; D. Lecic-Tosevski; D. Ajdukovic; T. Franciskovic; G.M. Galeazzi; A. Kucukalic; N. Morina; M. Popovski; M. Schützwohl; S. Priebe

    2012-01-01

    Purpose: The aim was to assess whether experiences of war trauma remain directly associated with suicidality in war affected communities when other risk factors are considered. Materials and methods: In the main sample 3313 participants from former Yugoslavia who experienced war trauma were recruite

  16. Active Affective Learning for Accelerated Schools.

    Science.gov (United States)

    Richardson, Robert B.

    This paper provides the groundwork for Active Affective Learning and teaching adapted to the needs of the disadvantaged, at-risk students served by the Accelerated Schools Movement. One of the "golden rules" for the practice of Accelerated Learning, according to psychiatrist Georgi Lozanov, has been to maintain an "up-beat" classroom presentation…

  17. Acknowledging the Affective in Higher Education

    Science.gov (United States)

    Beard, Colin; Clegg, Sue; Smith, Karen

    2007-01-01

    This article argues that we need richer conceptions of students as affective and embodied selves and a clearer theorisation of the role of emotion in educational encounters. These areas are currently under-researched and under-theorised in higher education. The first part of the article explores the literature on emotion. The second reports on a…

  18. View How Glaucoma May Affect Vision

    Science.gov (United States)

    ... inbox. Sign up View How Glaucoma May Affect Vision Normal Vision This is an example of normal vision. This is also an example of how someone ... gradual and often imperceptible failing of side (peripheral) vision. Intermediate Glaucoma As glaucoma progresses, the center of ...

  19. Processing speaker affect during spoken sentence comprehension

    NARCIS (Netherlands)

    van Leeuwen, A.R.; Quené, H.; van Berkum, J.J.A.

    2013-01-01

    Anne van Leeuwen Utrecht institute of Linguistics OTS, Utrecht University Processing speaker affect during spoken sentence comprehension We often smile (and frown) while we talk. Speakers use facial expression, posture and prosody to provide additional cues that signal speaker stance. Speaker stance

  20. Affective Learning and the Classroom Debate

    Science.gov (United States)

    Jagger, Suzy

    2013-01-01

    A commonly used teaching method to promote student engagement is the classroom debate. This study evaluates how affective characteristics, as defined in Bloom's taxonomy, were stimulated during debates that took place on a professional ethics module for first year computing undergraduates. The debates led to lively interactive group discussions…

  1. From affective blindsight to emotional consciousness.

    Science.gov (United States)

    Celeghin, Alessia; de Gelder, Beatrice; Tamietto, Marco

    2015-11-01

    Following destruction or denervation of the primary visual cortex (V1) cortical blindness ensues. Affective blindsight refers to the uncanny ability of such patients to respond correctly, or above chance level, to visual emotional expressions presented to their blind fields. Fifteen years after its original discovery, affective blindsight still fascinates neuroscientists and philosophers alike, as it offers a unique window on the vestigial properties of our visual system that, though present in the intact brain, tend to be unnoticed or even actively inhibited by conscious processes. Here we review available studies on affective blindsight with the intent to clarify its functional properties, neural bases and theoretical implications. Evidence converges on the role of subcortical structures of old evolutionary origin such as the superior colliculus, the pulvinar and the amygdala in mediating affective blindsight and nonconscious perception of emotions. We conclude that approaching consciousness, and its absence, from the vantage point of emotion processing may uncover important relations between the two phenomena, as consciousness may have evolved as an evolutionary specialization to interact with others and become aware of their social and emotional expressions.

  2. Phasic Affective Modulation of Semantic Priming

    Science.gov (United States)

    Topolinski, Sascha; Deutsch, Roland

    2013-01-01

    The present research demonstrates that very brief variations in affect, being around 1 s in length and changing from trial to trial independently from semantic relatedness of primes and targets, modulate the amount of semantic priming. Implementing consonant and dissonant chords (Experiments 1 and 5), naturalistic sounds (Experiment 2), and visual…

  3. Affect and Learning : a computational analysis

    NARCIS (Netherlands)

    Broekens, Douwe Joost

    2007-01-01

    In this thesis we have studied the influence of emotion on learning. We have used computational modelling techniques to do so, more specifically, the reinforcement learning paradigm. Emotion is modelled as artificial affect, a measure that denotes the positiveness versus negativeness of a situation

  4. Methods of Assessment for Affected Family Members

    Science.gov (United States)

    Orford, Jim; Templeton, Lorna; Velleman, Richard; Copello, Alex

    2010-01-01

    The article begins by making the point that a good assessment of the needs and circumstances of family members is important if previous neglect of affected family members is to be reversed. The methods we have used in research studies are then described. They include a lengthy semi-structured interview covering seven topic areas and standard…

  5. MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

    Science.gov (United States)

    Kostyrko, Kaja; Neuenschwander, Samuel; Junier, Thomas; Regamey, Alexandre; Iseli, Christian; Schmid-Siegert, Emanuel; Bosshard, Sandra; Majocchi, Stefano; Le Fourn, Valérie; Girod, Pierre-Alain; Xenarios, Ioannis; Mermod, Nicolas

    2017-02-01

    Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

  6. The spectrum and frequency of self-inflicted and host gene mutations produced by the transposon Ac in maize.

    Science.gov (United States)

    Huang, Jun T; Dooner, Hugo K

    2012-10-01

    The autonomous transposon Activator (Ac) is a powerful mutagen. Ac-induced mutations range from small footprints of host sequences to large rearrangements of transposon or host sequences. These mutations arise by different repair mechanisms of the double-strand break produced by Ac excision: footprints by nonhomologous end joining and rearrangements by various mechanisms, including DNA replication repair. Footprints greatly outnumber other mutations, masking them because they usually share a nonfunctional phenotype. To determine the spectrum and frequencies of host and self-mutations generated by Ac, we used an allele harboring Ac in the 5' untranslated region bronze (bz). In this system, simple excisions produce purple revertants, whereas deletions of host or transposon sequences produce stable bronze (bz-s) mutants. Internal and terminal deletions of Ac predominated among the 72 bz-s derivatives. Most internal deletions (52 of 54) behaved as nonautonomous Dissociation (Ds) elements. All nine terminal deletions or fractured Ac (fAc) elements had rearrangements of adjacent host sequences. Most Ds and fAc deletion junctions displayed microhomologies and contained filler DNA from nearby sequences, suggesting an origin by DNA repair synthesis followed by microhomology-mediated end joining. All mutations occurred more frequently in pollen, where one in 200 grains carried new Ds or fAc elements.

  7. The MRX Complex Ensures NHEJ Fidelity through Multiple Pathways Including Xrs2-FHA-Dependent Tel1 Activation.

    Directory of Open Access Journals (Sweden)

    Daichi Iwasaki

    2016-03-01

    Full Text Available Because DNA double-strand breaks (DSBs are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.

  8. Biochemical DSB-repair model for mammalian cells in G1 and early S phases of the cell cycle.

    Science.gov (United States)

    Taleei, Reza; Nikjoo, Hooshang

    2013-08-30

    The paper presents a model of double strand breaks (DSB) repair in G1 and early S phases of the cell cycle. The model is based on a plethora of published information on biochemical modification of DSB induced by ionizing radiation. So far, three main DSB repair pathways have been identified, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). During G1 and early S phases of the cell cycle, NHEJ and MMEJ repair pathways are activated dependent on the type of double strand breaks. Simple DSB are a substrate for NHEJ, while complex DSB and DSB in heterochromatin require further end processing. Repair of all DSB start with NHEJ presynaptic processes, and depending on the type of DSB pursue simple ligation, further end processing prior to ligation, or resection. Using law of mass action the model is translated into a mathematical formalism. The solution of the formalism provides the step by step and overall repair kinetics. The overall repair kinetics are compared with the published experimental measurements. Our calculations are in agreement with the experimental results and show that the complex types of DSBs are repaired with slow repair kinetics. The G1 and early S phase model could be employed to predict the kinetics of DSB repair for damage induced by high LET radiation.

  9. Nonreciprocal chromosomal translocations in renal cancer involve multiple DSBs and NHEJ associated with breakpoint inversion but not necessarily with transcription.

    Science.gov (United States)

    Ali, Hanif; Daser, Angelika; Dear, Paul; Wood, Henry; Rabbitts, Pamela; Rabbitts, Terence

    2013-04-01

    Chromosomal translocations and other abnormalities are central to the initiation of cancer in all cell types. Understanding the mechanism is therefore important to evaluate the evolution of cancer from the cancer initiating events to overt disease. Recent work has concentrated on model systems to develop an understanding of the molecular mechanisms of translocations but naturally occurring events are more ideal case studies since biological selection is absent from model systems. In solid tumours, nonreciprocal translocations are most commonly found, and accordingly we have investigated the recurrent nonreciprocal t(3;5) chromosomal translocations in renal carcinoma to better understand the mechanism of these naturally occurring translocations in cancer. Unexpectedly, the junctions of these translocations can be associated with site-specific, intrachromosomal inversion involving at least two double strand breaks (DSB) in cis and rejoining by nonhomologous end joining or micro-homology end joining. However, these translocations are not necessarily associated with transcribed regions questioning accessibility per se in controlling these events. In addition, intrachromosomal deletions also occur. We conclude these naturally occurring, nonreciprocal t(3;5) chromosomal translocations occur after complex and multiple unresolved intrachromosomal DSBs leading to aberrant joining with concurrent interstitial inversion and that clonal selection of cells is the critical element in cancer development emerging from a plethora of DSBs that may not always be pathogenic.

  10. More forks on the road to replication stress recovery

    Institute of Scientific and Technical Information of China (English)

    Chris Allen; Amanda K. Ashley; Robert Hromas; Jac A. Nickoloff

    2011-01-01

    High-fidelity replication of DNA, and its accurate segregation to daughter cells, is critical for maintaining genome stability and suppressing cancer. DNA replication forks are stalled by many DNA lesions, activating checkpoint proteins that stabilize stalled forks.Stalled forks may eventually collapse, producing a broken DNA end. Fork restart is typically mediated by proteins initially identified by their rotes in homologous recombination repair of DNA double-strand breaks (DSBs). In recent years, several proteins involved in DSB repair by non-homologous end joining (NHEJ) have been implicated in the replication stress response, including DNA-PKcs, Ku,DNA Ligase IV-XRCC4, Artemis, XLF and Metnase. It is currently unclear whether NHEJ proteins are involved in the replication stress response through indirect (signaling) roles, and/or direct roles involving DNA end joining. Additional complexity in the replication stress response centers around RPA, which undergoes significant post-translational modification after stress, and RAD52, a conserved HR protein whose role in DSB repair may have shifted to another protein in higher eukaryotes, such as BRCA2, but retained its rote in fork restart. Most cancer therapeutic strategies create DNA reputation stress. Thus, it is imperative to gain a better understanding of replication stress response proteins and pathways to improve cancer therapy.

  11. Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells.

    Science.gov (United States)

    Schwer, Bjoern; Wei, Pei-Chi; Chang, Amelia N; Kao, Jennifer; Du, Zhou; Meyers, Robin M; Alt, Frederick W

    2016-02-23

    High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.

  12. Assessing Affect after Mathematical Problem Solving Tasks: Validating the Chamberlin Affective Instrument for Mathematical Problem Solving

    Science.gov (United States)

    Chamberlin, Scott A.; Powers, Robert A.

    2013-01-01

    The focus of the article is the validation of an instrument to assess gifted students' affect after mathematical problem solving tasks. Participants were 225 students identified by their district as gifted in grades four to six. The Chamberlin Affective Instrument for Mathematical Problem Solving was used to assess feelings, emotions, and…

  13. Affective Development in Advanced Old Age: Analyses of Terminal Change in Positive and Negative Affect

    Science.gov (United States)

    Schilling, Oliver K.; Wahl, Hans-Werner; Wiegering, Sarah

    2013-01-01

    Late-life development of affect may unfold terminal changes that are driven more by end-of-life processes and not so much by time since birth. This study aimed to explore time-to-death-related effects in measures of affect in a sample of the very old. We used longitudinal data (2 measurement occasions: 2002 and 2003) from 140 deceased…

  14. Perfectionism, Performance, and State Positive Affect and Negative Affect after a Classroom Test

    Science.gov (United States)

    Flett, Gordon L.; Blankstein, Kirk R.; Hewitt, Paul L.

    2009-01-01

    The current study examined the associations among trait dimensions of perfectionism, test performance, and levels of positive and negative affect after taking a test. A sample of 92 female university students completed the Multidimensional Perfectionism Scale one week prior to an actual class test. Measures of positive affect and negative affect…

  15. Leader Affect and Leadership Effectiveness: How leader affective displays influence follower outcomes

    NARCIS (Netherlands)

    V.A. Visser (Victoria)

    2013-01-01

    textabstractThe aim of this dissertation is to uncover the relationship between leader affective displays and leadership effectiveness. Five empirical studies were conducted to test the influence of several leader affective displays on different follower outcomes that indicate leadership effectivene

  16. Affect, Reason, and Persuasion: Advertising Strategies That Predict Affective and Analytic-Cognitive Responses.

    Science.gov (United States)

    Chaudhuri, Arjun; Buck, Ross

    1995-01-01

    Develops and tests hypotheses concerning the relationship of specific advertising strategies to affective and analytic cognitive responses of the audience. Analyses undergraduate students' responses to 240 advertisements. Demonstrates that advertising strategy variables accounted substantially for the variance in affective and analytic cognition.…

  17. Multi-modal affect induction for affective brain-computer interfaces

    NARCIS (Netherlands)

    Mühl, C.; Broek, E.L. van den; Brouwer, A.M.; Nijboer, F.; Wouwe, N.C. van; Heylen, D.

    2011-01-01

    Reliable applications of affective brain-computer interfaces (aBCI) in realistic, multi-modal environments require a detailed understanding of the processes involved in emotions. To explore the modalityspecific nature of affective responses, we studied neurophysiological responses (i.e., EEG) of 24

  18. Contextualizing Mathematics Related Affect: Significance of Students' Individual and Social Level Affect in Finland and Chile

    Science.gov (United States)

    Tuohilampi, Laura

    2016-01-01

    Mathematics related affect turn from positive to negative during comprehensive school years worldwide. There is a clear need to find solutions to the problem. However, some gaps and problems appear in the methodologies and the common approaches used in the field. This article discusses five studies addressing affective development, challenges some…

  19. A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting

    Directory of Open Access Journals (Sweden)

    Zhao Yuguang

    2001-12-01

    Full Text Available Abstract Background Recent progress in insect transgenesis has been dramatic but existing transposon-based approaches are constrained by position effects and potential instability. Gene targeting would bring a number of benefits, however progress requires a better understanding of the mechanisms involved. Much can be learned in vitro since extrachromosomal recombination occurs at high frequency, facilitating the study of multiple events and the impact of structural changes among the recombining molecules. We have investigated homologous recombination in mosquito cells through restoration of luciferase activity from deleted substrates. The implications of this work for the construction of insect gene targeting vectors are discussed. Results We show that linear targeting vectors are significantly more efficient than circular ones and that recombination is stimulated by introducing double-strand breaks into, or near, the region of homology. Single-strand annealing represents a very efficient pathway but may not be feasible for targeting unbroken chromosomes. Using circular plasmids to mimic chromosomal targets, one-sided invasion appears to be the predominant pathway for homologous recombination. Non-homologous end joining reactions also occur and may be utilised in gene targeting if double-strand breaks are first introduced into the target site. Conclusions We describe a rapid, sensitive assay for extrachromosomal homologous recombination in mosquito cells. Variations in substrate topology suggest that single-strand annealing and one-sided invasion represent the predominant pathways, although non-homologous end joining reactions also occur. One-sided invasion of circular chromosomal mimics by linear vectors might therefore be used in vitro to investigate the design and efficiency of gene targeting strategies.

  20. The HSV-1 exonuclease, UL12, stimulates recombination by a single strand annealing mechanism.

    Science.gov (United States)

    Schumacher, April J; Mohni, Kareem N; Kan, Yinan; Hendrickson, Eric A; Stark, Jeremy M; Weller, Sandra K

    2012-01-01

    Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5'-to-3' exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.