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Sample records for break repair complex

  1. Structure of the Rad50 DNA double-strand break repair protein in complex with DNA.

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    Rojowska, Anna; Lammens, Katja; Seifert, Florian U; Direnberger, Carolin; Feldmann, Heidi; Hopfner, Karl-Peter

    2014-12-01

    The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50(NBD) (nucleotide-binding domain) in complex with Mre11(HLH) (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state. © 2014 The Authors.

  2. The processing of double-stranded DNA breaks for recombinational repair by helicase-nuclease complexes.

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    Yeeles, Joseph T P; Dillingham, Mark S

    2010-03-02

    Double-stranded DNA breaks are prepared for recombinational repair by nucleolytic digestion to form single-stranded DNA overhangs that are substrates for RecA/Rad51-mediated strand exchange. This processing can be achieved through the activities of multiple helicases and nucleases. In bacteria, the function is mainly provided by a stable multi-protein complex of which there are two structural classes; AddAB- and RecBCD-type enzymes. These helicase-nucleases are of special interest with respect to DNA helicase mechanism because they are exceptionally powerful DNA translocation motors, and because they serve as model systems for both single molecule studies and for understanding how DNA helicases can be coupled to other protein machinery. This review discusses recent developments in our understanding of the AddAB and RecBCD complexes, focussing on their distinctive strategies for processing DNA ends. We also discuss the extent to which bacterial DNA end resection mechanisms may parallel those used in eukaryotic cells. (c) 2010 Elsevier B.V. All rights reserved.

  3. Role for the mammalian Swi5-Sfr1 complex in DNA strand break repair through homologous recombination.

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    Yufuko Akamatsu

    2010-10-01

    Full Text Available In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR, a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5(-/- and Sfr1(-/- embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5(-/- and Sfr1(-/- cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5(-/- and Sfr1(-/- cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks.

  4. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

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    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.

  5. ZTF-8 interacts with the 9-1-1 complex and is required for DNA damage response and double-strand break repair in the C. elegans germline.

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    Hyun-Min Kim

    2014-10-01

    Full Text Available Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR and DSB repair (DSBR within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.

  6. Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease.

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    Wenjian Ma

    2011-04-01

    Full Text Available DNA double-strand breaks (DSBs are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs with 3'-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3' blocked single-strand breaks following the creation of abasic (AP sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3'-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.

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

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    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. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  8. MTE1 Functions with MPH1 in Double-Strand Break Repair.

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    Yimit, Askar; Kim, TaeHyung; Anand, Ranjith P; Meister, Sarah; Ou, Jiongwen; Haber, James E; Zhang, Zhaolei; Brown, Grant W

    2016-05-01

    Double-strand DNA breaks occur upon exposure of cells to ionizing radiation and certain chemical agents or indirectly through replication fork collapse at DNA damage sites. If left unrepaired, double-strand breaks can cause genome instability and cell death, and their repair can result in loss of heterozygosity. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination relocalize into discrete nuclear foci. We identified 29 proteins that colocalize with recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase gene MPH1 is absent. Mte1 and Mph1 form a complex and are recruited to double-strand breaks in vivo in a mutually dependent manner. MTE1 is important for resolution of Rad52 foci during double-strand break repair and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair. Copyright © 2016 by the Genetics Society of America.

  9. RNA-directed repair of DNA double-strand breaks.

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    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. DEK is required for homologous recombination repair of DNA breaks

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    Smith, Eric A; Gole, Boris; Willis, Nicholas A

    2017-01-01

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency pheno......DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK......-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout...

  11. Double strand break repair functions of histone H2AX

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    Scully, Ralph, E-mail: rscully@bidmc.harvard.edu; Xie, Anyong

    2013-10-15

    Chromosomal double strand breaks provoke an extensive reaction in neighboring chromatin, characterized by phosphorylation of histone H2AX on serine 139 of its C-terminal tail (to form “γH2AX”). The γH2AX response contributes to the repair of double strand breaks encountered in a variety of different contexts, including those induced by ionizing radiation, physiologically programmed breaks that characterize normal immune cell development and the pathological exposure of DNA ends triggered by telomere dysfunction. γH2AX also participates in the evolutionarily conserved process of sister chromatid recombination, a homologous recombination pathway involved in the suppression of genomic instability during DNA replication and directly implicated in tumor suppression. At a biochemical level, the γH2AX response provides a compelling example of how the “histone code” is adapted to the regulation of double strand break repair. Here, we review progress in research aimed at understanding how γH2AX contributes to double strand break repair in mammalian cells.

  12. Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination.

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    Orta, Manuel Luis; Pastor, Nuria; Burgos-Morón, Estefanía; Domínguez, Inmaculada; Calderón-Montaño, José Manuel; Huertas Castaño, Carlos; López-Lázaro, Miguel; Helleday, Thomas; Mateos, Santiago

    2017-09-01

    Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It's well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. A role for small RNAs in DNA double-strand break repair

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    Wei, W.; Ba, Z.; Wu, Y.

    2012-01-01

    Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells....... We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, di...

  14. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

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    Gao, Min; Wei, Wei; Li, Ming Hua

    2014-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute...... cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB...

  15. TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats

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    Valentine Mosbach

    2018-02-01

    Full Text Available Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington’s disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.

  16. Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.

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    Slawomir Kumala

    Full Text Available To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.

  17. Genetics of x-ray induced double strand break repair in saccharomyces cerevisiae

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    Budd, M.E.

    1982-07-01

    The possible fates of x-ray-induced double-strand breaks in Saccharomyces cerevisiae were examined. One possible pathway which breaks can follow, the repair pathway, was studied by assaying strains with mutations in the RAD51, RAD54, and RAD57 loci for double-strand break repair. In order of increasing radiation sensitivity one finds: rad57-1(23/sup 0/)> rad51-1(30/sup 0/)> rad54-3(36/sup 0/). At 36/sup 0/, rad54-3 cells cannot repair double-strand breaks, while 23/sup 0/, they can. Strains with the rad57-1 mutation can rejoin broken chromosomes at both temperatures. However, the low survival at 36/sup 0/ shows that the assay is not distinguishing large DNA fragments which allow cell survival from those which cause cell death. A rad51-1 strain could also rejoin broken chromosomes, and was thus capable of incomplete repair. The data can be explained with the hypothesis that rad54-3 cells are blocked in an early step of repair, while rad51-1 and rad57-1 strains are blocked in a later step of repair. The fate of double-strand breaks when they are left unrepaired was investigated with the rad54-3 mutation. If breaks are prevented from entering the RAD54 repair pathway they become uncommitted lesions. These lesions are repaired slower than the original breaks. One possible fate for an uncommitted lesion is conversion into a fixed lesion, which is likely to be an unrepairable or misrepaired double-strand break. The presence of protein synthesis after irradiation increases the probability that a break will enter the repair pathway. Evidence shows that increased probability of repair results from enhanced synthesis of repair proteins shortly after radiation. (ERB)

  18. Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex

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    Géli, Vincent; Lisby, Michael

    2015-01-01

    The nuclear pore complex (NPC) is emerging as a center for recruitment of a class of "difficult to repair" lesions such as double-strand breaks without a repair template and eroded telomeres in telomerase-deficient cells. In addition to such pathological situations, a recent study by Su and colle......The nuclear pore complex (NPC) is emerging as a center for recruitment of a class of "difficult to repair" lesions such as double-strand breaks without a repair template and eroded telomeres in telomerase-deficient cells. In addition to such pathological situations, a recent study by Su...

  19. Differential usage of alternative pathways of double-strand break repair in Drosophila.

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    Preston, Christine R; Flores, Carlos C; Engels, William R

    2006-02-01

    Double-strand DNA breaks can be repaired by any of several alternative mechanisms that differ greatly in the nature of the final repaired products. We used a reporter construct, designated "Repair reporter 3" (Rr3), to measure the relative usage of these pathways in Drosophila germ cells. The method works by creating a double-strand break at a specific location such that expression of the red fluorescent protein, DsRed, in the next generation can be used to infer the frequency at which each pathway was used. A key feature of this approach is that most data come from phenotypic scoring, thus allowing large sample sizes and considerable precision in measurements. Specifically, we measured the proportion of breaks repaired by (1) conversion repair, (2) nonhomologous end joining (NHEJ), or (3) single-strand annealing (SSA). For conversion repair, the frequency of mitotic crossing over in the germ line indicates the relative prevalence of repair by double Holliday junction (DHJ) formation vs. the synthesis-dependent strand annealing (SDSA) pathway. We used this method to show that breaks occurring early in germ-line development were much more frequently repaired via single-strand annealing and much less likely to be repaired by end joining compared with identical breaks occurring later in development. Conversion repair was relatively rare when breaks were made either very early or very late in development, but was much more frequent in between. Significantly, the changes in relative usage occurred in a compensatory fashion, such that an increase in one pathway was accompanied by decreases in others. This negative correlation is interpreted to mean that the pathways for double-strand break repair compete with each other to handle a given breakage event.

  20. Adriamycin does not affect the repair of X-ray induced DNA single strand breaks

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    Cantoni, O.; Sestili, P.; Cattabeni, F.

    1985-06-01

    The ability of the antitumor antibiotic adriamycin (Ad) to inhibit the rejoining of DNA single strand breaks produced by X-rays was investigated in cultured cells. Chinese hamster ovary cells were given 400 rad and were allowed to repair in the presence or absence of Ad for 60 min at 37degC. The drug did not affect the ability of cells to repair DNA breaks and residual breaks found after the repair period were attributed to those induced by Ad alone. (author). 16 refs.

  1. Complex networks under dynamic repair model

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    Chaoqi, Fu; Ying, Wang; Kun, Zhao; Yangjun, Gao

    2018-01-01

    Invulnerability is not the only factor of importance when considering complex networks' security. It is also critical to have an effective and reasonable repair strategy. Existing research on network repair is confined to the static model. The dynamic model makes better use of the redundant capacity of repaired nodes and repairs the damaged network more efficiently than the static model; however, the dynamic repair model is complex and polytropic. In this paper, we construct a dynamic repair model and systematically describe the energy-transfer relationships between nodes in the repair process of the failure network. Nodes are divided into three types, corresponding to three structures. We find that the strong coupling structure is responsible for secondary failure of the repaired nodes and propose an algorithm that can select the most suitable targets (nodes or links) to repair the failure network with minimal cost. Two types of repair strategies are identified, with different effects under the two energy-transfer rules. The research results enable a more flexible approach to network repair.

  2. The transcription fidelity factor GreA impedes DNA break repair.

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    Sivaramakrishnan, Priya; Sepúlveda, Leonardo A; Halliday, Jennifer A; Liu, Jingjing; Núñez, María Angélica Bravo; Golding, Ido; Rosenberg, Susan M; Herman, Christophe

    2017-10-12

    Homologous recombination repairs DNA double-strand breaks and must function even on actively transcribed DNA. Because break repair prevents chromosome loss, the completion of repair is expected to outweigh the transcription of broken templates. However, the interplay between DNA break repair and transcription processivity is unclear. Here we show that the transcription factor GreA inhibits break repair in Escherichia coli. GreA restarts backtracked RNA polymerase and hence promotes transcription fidelity. We report that removal of GreA results in markedly enhanced break repair via the classic RecBCD-RecA pathway. Using a deep-sequencing method to measure chromosomal exonucleolytic degradation, we demonstrate that the absence of GreA limits RecBCD-mediated resection. Our findings suggest that increased RNA polymerase backtracking promotes break repair by instigating RecA loading by RecBCD, without the influence of canonical Chi signals. The idea that backtracked RNA polymerase can stimulate recombination presents a DNA transaction conundrum: a transcription fidelity factor that compromises genomic integrity.

  3. Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair.

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    Ohle, Corina; Tesorero, Rafael; Schermann, Géza; Dobrev, Nikolay; Sinning, Irmgard; Fischer, Tamás

    2016-11-03

    RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. SAW1 is required for SDSA double-strand break repair in S. cerevisiae.

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    Diamante, Graciel; Phan, Claire; Celis, Angie S; Krueger, Jonas; Kelson, Eric P; Fischhaber, Paula L

    2014-03-14

    SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Linking Structure and Function for the DNA Repair Complex Mre11-Rad50-Nbs1

    NARCIS (Netherlands)

    E. Kinoshita (Eri)

    2014-01-01

    markdownabstract__Abstract__ Repair of DNA damage is an essential process in all cells and an important mechanism to avoid cancer development in animals. The repair of DNA double strand breaks (DSB) requires many component proteins including the Mre11-Rad50-Nbs1 (MRN) complex that serves

  6. Structure and Stability of ERCC1-XPF DNA Repair Complexes

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    Faridounnia, M.

    2015-01-01

    Understanding DNA repair pathways such as Nucleotide Excision Repair, Double Strand Break repair and Interstrand Cross-Link repair is of basic interest for understanding fundamental cellular processes. It also forms the basis for understanding molecular details of diseases when defects occur in

  7. Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre

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    Lisby, M.; Mortensen, Uffe Hasbro; Rothstein, R.

    2003-01-01

    DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in ...

  8. Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

    NARCIS (Netherlands)

    H.B. Beverloo (Berna); R.D. Johnson (Roger); M. Jasin (Maria); R. Kanaar (Roland); J.H.J. Hoeijmakers (Jan); M.L.G. Dronkert (Mies)

    2000-01-01

    textabstractCells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we

  9. Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids.

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    Ahmed, E.A.; Boer, P. de; Philippens, M.E.P.; Kal, H.B.; Rooij, D.G. de

    2010-01-01

    The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of

  10. Non Random Distribution of DMD Deletion Breakpoints and Implication of Double Strand Breaks Repair and Replication Error Repair Mechanisms.

    Science.gov (United States)

    Marey, Isabelle; Ben Yaou, Rabah; Deburgrave, Nathalie; Vasson, Aurélie; Nectoux, Juliette; Leturcq, France; Eymard, Bruno; Laforet, Pascal; Behin, Anthony; Stojkovic, Tanya; Mayer, Michèle; Tiffreau, Vincent; Desguerre, Isabelle; Boyer, François Constant; Nadaj-Pakleza, Aleksandra; Ferrer, Xavier; Wahbi, Karim; Becane, Henri-Marc; Claustres, Mireille; Chelly, Jamel; Cossee, Mireille

    2016-05-27

    Dystrophinopathies are mostly caused by copy number variations, especially deletions, in the dystrophin gene (DMD). Despite the large size of the gene, deletions do not occur randomly but mainly in two hot spots, the main one involving exons 45 to 55. The underlying mechanisms are complex and implicate two main mechanisms: Non-homologous end joining (NHEJ) and micro-homology mediated replication-dependent recombination (MMRDR). Our goals were to assess the distribution of intronic breakpoints (BPs) in the genomic sequence of the main hot spot of deletions within DMD gene and to search for specific sequences at or near to BPs that might promote BP occurrence or be associated with DNA break repair. Using comparative genomic hybridization microarray, 57 deletions within the intron 44 to 55 region were mapped. Moreover, 21 junction fragments were sequenced to search for specific sequences. Non-randomly distributed BPs were found in introns 44, 47, 48, 49 and 53 and 50% of BPs clustered within genomic regions of less than 700bp. Repeated elements (REs), known to promote gene rearrangement via several mechanisms, were present in the vicinity of 90% of clustered BPs and less frequently (72%) close to scattered BPs, illustrating the important role of such elements in the occurrence of DMD deletions. Palindromic and TTTAAA sequences, which also promote DNA instability, were identified at fragment junctions in 20% and 5% of cases, respectively. Micro-homologies (76%) and insertions or deletions of small sequences were frequently found at BP junctions. Our results illustrate, in a large series of patients, the important role of RE and other genomic features in DNA breaks, and the involvement of different mechanisms in DMD gene deletions: Mainly replication error repair mechanisms, but also NHEJ and potentially aberrant firing of replication origins. A combination of these mechanisms may also be possible.

  11. Genetic and environmental influence on DNA strand break repair: a twin study

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander

    2013-01-01

    Accumulation of DNA damage deriving from exogenous and endogenous sources has significant consequences for cellular survival, and is implicated in aging, cancer, and neurological diseases. Different DNA repair pathways have evolved in order to maintain genomic stability. Genetic and environmental......-strand breaks), and some of the most hazardous lesions (DNA double-strand breaks). DNA damage signaling response (Gamma-H2AX signaling), relative amount of endogenous damage, and DNA-strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104...

  12. IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

    Science.gov (United States)

    Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

    2017-03-01

    Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

  13. Complex networks repair strategies: Dynamic models

    Science.gov (United States)

    Fu, Chaoqi; Wang, Ying; Gao, Yangjun; Wang, Xiaoyang

    2017-09-01

    Network repair strategies are tactical methods that restore the efficiency of damaged networks; however, unreasonable repair strategies not only waste resources, they are also ineffective for network recovery. Most extant research on network repair focuses on static networks, but results and findings on static networks cannot be applied to evolutionary dynamic networks because, in dynamic models, complex network repair has completely different characteristics. For instance, repaired nodes face more severe challenges, and require strategic repair methods in order to have a significant effect. In this study, we propose the Shell Repair Strategy (SRS) to minimize the risk of secondary node failures due to the cascading effect. Our proposed method includes the identification of a set of vital nodes that have a significant impact on network repair and defense. Our identification of these vital nodes reduces the number of switching nodes that face the risk of secondary failures during the dynamic repair process. This is positively correlated with the size of the average degree 〈 k 〉 and enhances network invulnerability.

  14. Cell cycle-regulated centers of DNA double-strand break repair

    DEFF Research Database (Denmark)

    Lisby, Michael; Antúnez de Mayolo, Adriana; Mortensen, Uffe H

    2003-01-01

    In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks. We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker. In response to DNA damage, Rad52 redistributes itself and forms...... foci specifically during S phase. We have shown previously that Rad52 foci are centers of DNA repair where multiple DNA double-strand breaks colocalize. Here we report a correlation between the timing of Rad52 focus formation and modification of the Rad52 protein. In addition, we show that the two ends...... of a double-strand break are held tightly together in the majority of cells. Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair....

  15. Efficient repair of DNA breaks in Drosophila: evidence for single-strand annealing and competition with other repair pathways.

    Science.gov (United States)

    Preston, Christine R; Engels, William; Flores, Carlos

    2002-06-01

    We show evidence that DNA double-strand breaks induced in the Drosophila germ line can be repaired very efficiently by the single-strand annealing (SSA) mechanism. A double-strand break was made between two copies of a 1290-bp direct repeat by mobilizing a P transposon. In >80% of the progeny that acquired this chromosome, repair resulted in loss of the P element and loss of one copy of the repeat, as observed in SSA. The frequency of this repair was much greater than seen for gene conversion using an allelic template, which is only approximately 7%. A similar structure, but with a smaller duplication of only 158 bp, also yielded SSA-like repair events, but at a reduced frequency, and gave rise to some products by repair pathways other than SSA. The 1290-bp repeats carried two sequence polymorphisms that were examined in the products. The allele nearest to a nick in the putative heteroduplex intermediate was lost most often. This bias is predicted by the SSA model, although other models could account for it. We conclude that SSA is the preferred repair pathway in Drosophila for DNA breaks between sequence repeats, and it competes with gene conversion by the synthesis-dependent strand annealing (SDSA) pathway.

  16. Numt-mediated double-strand break repair mitigates deletions during primate genome evolution.

    Directory of Open Access Journals (Sweden)

    Einat Hazkani-Covo

    2008-10-01

    Full Text Available Non-homologous end joining (NHEJ is the major mechanism of double-strand break repair (DSBR in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs. Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler

  17. Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander

    2013-01-01

    Exogenous and endogenous damage to DNA is constantly challenging the stability of our genome. This DNA damage increase the frequency of errors in DNA replication, thus causing point mutations or chromosomal rearrangements and has been implicated in aging, cancer, and neurodegenerative diseases....... Therefore, efficient DNA repair is vital for the maintenance of genome stability. The general notion has been that DNA repair capacity decreases with age although there are conflicting results. Here, we focused on potential age-associated changes in DNA damage response and the capacities of repairing DNA...... single-strand breaks (SSBs) and double-strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ-H2AX response, and DSB repair capacity...

  18. Meiotic versus Mitotic Recombination: Two Different Routes for Double-Strand Break Repair

    Science.gov (United States)

    Andersen, Sabrina L.; Sekelsky, Jeff

    2011-01-01

    Summary Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, and it is becoming increasing clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically proliferating cells; rather, the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires crossovers) than mitotic recombination (repair of DNA damage, which typically generates non-crossovers). PMID:20967781

  19. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    Science.gov (United States)

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-06-01

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  20. DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair.

    Science.gov (United States)

    Sibanda, Bancinyane L; Chirgadze, Dimitri Y; Ascher, David B; Blundell, Tom L

    2017-02-03

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128-amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination. Copyright © 2017, American Association for the Advancement of Science.

  1. Differential requirement for SUB1 in chromosomal and plasmid double-strand DNA break repair.

    Directory of Open Access Journals (Sweden)

    Lijian Yu

    Full Text Available Non homologous end joining (NHEJ is an important process that repairs double strand DNA breaks (DSBs in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

  2. Homology Requirements and Competition between Gene Conversion and Break-Induced Replication during Double-Strand Break Repair.

    Science.gov (United States)

    Mehta, Anuja; Beach, Annette; Haber, James E

    2017-02-02

    Saccharomyces cerevisiae mating-type switching is initiated by a double-strand break (DSB) at MATa, leaving one cut end perfectly homologous to the HMLα donor, while the second end must be processed to remove a non-homologous tail before completing repair by gene conversion (GC). When homology at the matched end is ≤150 bp, efficient repair depends on the recombination enhancer, which tethers HMLα near the DSB. Thus, homology shorter than an apparent minimum efficient processing segment can be rescued by tethering the donor near the break. When homology at the second end is ≤150 bp, second-end capture becomes inefficient and repair shifts from GC to break-induced replication (BIR). But when pol32 or pif1 mutants block BIR, GC increases 3-fold, indicating that the steps blocked by these mutations are reversible. With short second-end homology, absence of the RecQ helicase Sgs1 promotes gene conversion, whereas deletion of the FANCM-related Mph1 helicase promotes BIR. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Expansions and contractions in a tandem repeat induced by double-strand break repair.

    Science.gov (United States)

    Pâques, F; Leung, W Y; Haber, J E

    1998-04-01

    Repair of a double-strand break (DSB) in yeast can induce very frequent expansions and contractions in a tandem array of 375-bp repeats. These results strongly suggest that DSB repair can be a major source of amplification of tandemly repeated sequences. Most of the DSB repair events are not associated with crossover. Rearrangements appear in 50% of these repaired recipient molecules. In contrast, the donor template nearly always remains unchanged. Among the rare crossover events, similar rearrangements are found. These results cannot readily be explained by the gap repair model of Szostak et al. (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) but can be explained by synthesis-dependent strand annealing (SDSA) models that allow for crossover. Support for SDSA models is provided by a demonstration that a single DSB repair event can use two donor templates located on two different chromosomes.

  4. DEK is required for homologous recombination repair of DNA breaks

    DEFF Research Database (Denmark)

    Smith, Eric A; Gole, Boris; Willis, Nicholas A

    2017-01-01

    mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK...

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

    OpenAIRE

    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. ...

  6. RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks

    DEFF Research Database (Denmark)

    Lu, Huiming; Shamanna, Raghavendra A; Keijzers, Guido

    2016-01-01

    The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR...... interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4's helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4's......). Depletion of RECQL4 severely reduces HR-mediated repair and 5' end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly...

  7. Breaking bad: The mutagenic effect of DNA repair

    Science.gov (United States)

    2015-01-01

    Species survival depends on the faithful replication of genetic information, which is continually monitored and maintained by DNA repair pathways thatcorrect replication errors and the thousands of lesions that arise daily from the inherent chemical lability of DNA and the effects of genotoxic agents. Nonetheless,neutrally evolving DNA (not under purifying selection) accumulates base substitutions with time (the neutral mutation rate). Thus, repair processes are not 100% efficient. The neutral mutation rate varies both between and within chromosomes. For example it is 10 – 50 fold higher at CpGsthan at non-CpG positions. Interestingly, the neutral mutation rate at non-CpG sites is positively correlated with CpG content. Althoughthe basis of this correlation was not immediately apparent,some bioinformatic results were consistent with the induction of non-CpGmutations byDNA repairat flanking CpG sites. Recent studies with a model system showed that in vivo repair of preformed lesions (mismatches, abasic sites, single stranded nicks) can in factinduce mutations in flanking DNA. Mismatch repair (MMR) is an essential component for repair-induced mutations, which can occur as distant as 5 kb from the introduced lesions. Most, but not all, mutations involved the C of TpCpN (G of NpGpA) which is the target sequence of the C-preferringsingle-stranded DNA specific APOBEC deaminases. APOBEC-mediated mutations are not limited to our model system: Recent studies by others showed that some tumors harbor mutations with the same signature, as can intermediates in RNA-guided endonuclease-mediated genome editing. APOBEC deaminases participate in normal physiological functions such as generating mutations that inactivate viruses or endogenous retrotransposons, or that enhance immunoglobulin diversity in B cells. The recruitment of normally physiological errorprone processes during DNA repairwould have important implications for disease, aging and evolution. This perspective briefly

  8. 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. PMID:26437586

  9. Impact of charged particle exposure on homologous DNA double-strand break repair in human blood-derived cells

    Directory of Open Access Journals (Sweden)

    Melanie eRall

    2015-11-01

    Full Text Available Ionizing radiation generates DNA double-strand breaks (DSB which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC, potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL, particularly regarding homologous DSB repair (HR. Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET in HSPC versus PBL. For higher LET, 53BP1 foci kinetics were similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose-dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment.

  10. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

    DEFF Research Database (Denmark)

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs...

  11. Repair and genetic consequences of DNA double strand breaks during animal development

    NARCIS (Netherlands)

    Lemmens, Bennie Benjamin Lodewijk Gerardus

    2014-01-01

    The genetic code of life is stored in DNA molecules that consist of two parallel strands of coupled nucleotides that form a DNA double helix. One of the most deleterious forms of DNA damage is a DNA double-strand break (DSB) in which both strands of the helix are broken. When not repaired adequately

  12. Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants.

    Directory of Open Access Journals (Sweden)

    Bret R Adams

    2010-04-01

    Full Text Available The DNA double-strand break (DSB is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ is dominant. We have characterized the DNA damage response (DDR and quality of DNA double-strand break (DSB repair in human embryonic stem cells (hESCs, and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR, showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ] in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

  13. Regulation of repair pathway choice at two-ended DNA double-strand breaks.

    Science.gov (United States)

    Shibata, Atsushi

    2017-10-01

    A DNA double-strand break (DSB) is considered to be a critical DNA lesion because its misrepair can cause severe mutations, such as deletions or chromosomal translocations. For the precise repair of DSBs, the repair pathway that is optimal for the particular circumstance needs to be selected. Non-homologous end joining (NHEJ) functions in G1/S/G2 phase, while homologous recombination (HR) becomes active only in S/G2 phase after DNA replication. DSB end structure is another factor affecting the repair pathway. For example, one-ended DSBs in S phase are mainly repaired by HR due to the lack of a partner DSB end for NHEJ. In contrast, two-ended DSBs, which are mainly induced by ionizing radiation, are repaired by either NHEJ or HR in G2 cells. Under the current model in terms of DSB repair pathway usage in G2 phase, NHEJ repairs ∼70% of two-ended DSBs, whereas HR repairs only ∼30%. Recent studies propose that NHEJ factors can bind all the DSB ends and are then either used to progress that pathway of DSB repair, or the repair proceeds by HR. In addition, molecular regulation by BRCA1 and 53BP1 has also been proposed. At DSB sites, BRCA1 functions to alleviate the 53BP1 barrier to resection by promoting 53BP1 dephosphorylation, followed by RIF1 release and 53BP1 repositioning. This timely 53BP1 repositioning may be important for the establishment of a chromatin environment that promotes the recruitment of EXO1 for resection in HR. This review summarizes current knowledge on factors regulating DSB repair pathway choice in terms of spatiotemporal regulation by focusing on the repair events at two-ended DSBs in G2 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. The Heterochromatic Barrier to DNA Double Strand Break Repair: How to Get the Entry Visa

    Directory of Open Access Journals (Sweden)

    Aaron A. Goodarzi

    2012-09-01

    Full Text Available Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ and homologous recombination (HR represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC and highly compacted, transcriptionally inert, heterochromatin (HC, although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM, is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair.

  15. Local Activity Principle:. the Cause of Complexity and Symmetry Breaking

    Science.gov (United States)

    Mainzer, Klaus

    2013-01-01

    The principle of local activity is precisely the missing concept to explain the emergence of complex patterns in a homogeneous medium. Leon O. Chua discovered and defined this principle in the theory of nonlinear electronic circuits in a mathematically rigorous way. The local principle can be generalized and proven at least for the class of nonlinear reaction-diffusion systems in physics, chemistry, biology and brain research. Recently, it was realized by memristors for nanoelectronic device applications in technical brains. In general, the emergence of complex patterns and structures is explained by symmetry breaking in homogeneous media. The principle of local activity is the cause of symmetry breaking in homogeneous media. We argue that the principle of local activity is really fundamental in science and can even be identified in quantum cosmology as symmetry breaking of local gauge symmetries generating the complexity of matter and forces in our universe. Finally, we consider applications in economic, financial, and social systems with the emergence of equilibrium states, symmetry breaking at critical points of phase transitions and risky acting at the edge of chaos. In any case, the driving causes of symmetry breaking and the emergence of complexity are locally active elements, cells, units, or agents.

  16. DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance.

    Science.gov (United States)

    Rother, Magdalena B; van Attikum, Haico

    2017-10-05

    Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Authors.

  17. The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.

    Science.gov (United States)

    Bailly, Aymeric P; Freeman, Alasdair; Hall, Julie; Déclais, Anne-Cécile; Alpi, Arno; Lilley, David M J; Ahmed, Shawn; Gartner, Anton

    2010-07-15

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

  18. The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Aymeric P Bailly

    2010-07-01

    Full Text Available DNA double-strand breaks (DSBs can be repaired by homologous recombination (HR, which can involve Holliday junction (HJ intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

  19. SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

    DEFF Research Database (Denmark)

    McCord, Ronald A; Michishita, Eriko; Hong, Tao

    2009-01-01

    The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA......-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor...

  20. OsRAD51C Is Essential for Double Strand Break Repair in Rice Meiosis

    Directory of Open Access Journals (Sweden)

    Ding eTang

    2014-05-01

    Full Text Available RAD51C is one of the RAD51 paralogs that plays an important role in DNA double-strand break repair by homologous recombination. Here, we identified and characterized OsRAD51C, the rice homolog of human RAD51C. The Osrad51c mutant plant is normal in vegetative growth but exhibits complete male and female sterility. Cytological investigation revealed that homologous pairing and synapsis were severely disrupted. Massive chromosome fragmentation occurred during metaphase I in Osrad51c meiocytes, and was fully suppressed by the CRC1 mutation. Immunofluorescence analysis showed that OsRAD51C localized onto the chromosomes from leptotene to early pachytene during prophase I, and that normal loading of OsRAD51C was dependent on OsREC8, PAIR2, and PAIR3. Additionally, ZEP1 did not localize properly in Osrad51c, indicating that OsRAD51C is required for synaptonemal complex assembly. Our study also provided evidence in support of a functional divergence in RAD51C among organisms.

  1. Granulocytes affect double-strand break repair assays in primary human lymphocytes.

    Science.gov (United States)

    Lacoste, Sandrine; Bhatia, Ravi; Bhatia, Smita; O'Connor, Timothy R

    2014-01-01

    Patients who develop therapy-related myelodysplasia/acute myeloid leukemia after autologous-hematopoietic stem cell (aHCT) transplant show lower expression levels of DNA repair genes in their pre-aHCT CD34+ cells. To investigate whether this leads to functional differences in DNA repair abilities measurable in patients, we adapted two plasmid-based host-cell reactivation assays for use in primary lymphocytes. Prior to applying these assays to patients who underwent aHCT, we wanted first to verify whether sample preparation affected repair measurements, as patient samples were simply depleted of erythrocytes (with hetastarch) prior to freezing, which is not the classical way to prepare lymphocytes prior to DNA repair experiments (with a density gradient). We show here that lymphocytes from healthy donors freshly prepared with hetastarch show systematically a higher level of double-strand break repair as compared to when prepared with a density gradient, but that most of this difference disappears after samples were frozen. Several observations points to granulocytes as the source for this effect of sample preparation on repair: 1) removal of granulocytes makes the effect disappear, 2) DSB repair measurements for the same individual correlate to the percentage of granulocytes in the sample and 3) nucleofection in presence of granulocytes increases the level of reactive oxygen species (ROS) in neighboring lymphocytes in a dose-dependent manner (R2 of 0.95). These results indicate that co-purified granulocytes, possibly through the release of ROS at time of transfection, can lead to an enhanced repair in lymphocytes that obfuscates any evaluation of inter individual differences in repair as measured by host-cell reactivation. As a result, hetastarch-prepared samples are likely unsuitable for the assessment of DSB repair in primary cells with that type of assay. Granulocyte contamination that exists after a density gradient preparation, although much more limited, could

  2. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

    DEFF Research Database (Denmark)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

    2016-01-01

    RNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian......Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose...... RAD52 facilitates repair of collapsed DNA replication forks in cancer cells....

  3. SAW1 is Required for SDSA Double-Strand Break Repair in S. cerevisiae

    OpenAIRE

    Diamante, Graciel; Phan, Claire; Celis, Angie S.; Krueger, Jonas; Kelson, Eric P.; Fischhaber, Paula L.

    2014-01-01

    SAW1 , coding for Saw1, is required for single-strand annealing (SSA) DNA Double-strand Break (DSB) Repair in S. cerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized...

  4. Atypical Role for PhoU in Mutagenic Break Repair under Stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Janet L Gibson

    Full Text Available Mechanisms of mutagenesis activated by stress responses drive pathogen/host adaptation, antibiotic and anti-fungal-drug resistance, and perhaps much of evolution generally. In Escherichia coli, repair of double-strand breaks (DSBs by homologous recombination is high fidelity in unstressed cells, but switches to a mutagenic mode using error-prone DNA polymerases when the both the SOS and general (σS stress responses are activated. Additionally, the σE response promotes spontaneous DNA breakage that leads to mutagenic break repair (MBR. We identified the regulatory protein PhoU in a genetic screen for functions required for MBR. PhoU negatively regulates the phosphate-transport and utilization (Pho regulon when phosphate is in excess, including the PstB and PstC subunits of the phosphate-specific ABC transporter PstSCAB. Here, we characterize the PhoU mutation-promoting role. First, some mutations that affect phosphate transport and Pho transcriptional regulation decrease mutagenesis. Second, the mutagenesis and regulon-expression phenotypes do not correspond, revealing an apparent new function(s for PhoU. Third, the PhoU mutagenic role is not via activation of the σS, SOS or σE responses, because mutations (or DSBs that restore mutagenesis to cells defective in these stress responses do not restore mutagenesis to phoU cells. Fourth, the mutagenesis defect in phoU-mutant cells is partially restored by deletion of arcA, a gene normally repressed by PhoU, implying that a gene(s repressed by ArcA promotes mutagenic break repair. The data show a new role for PhoU in regulation, and a new regulatory branch of the stress-response signaling web that activates mutagenic break repair in E. coli.

  5. BRCA2 and the DNA Double-Strand Break Repair Machinery

    National Research Council Canada - National Science Library

    Chen, Phang-Lang

    2000-01-01

    The overall goal of my grant proposal is to test the model, BRCA2 modulates the early steps of repair mediated by the Rad5O nuclease complex, and the later stages catalyzed by the Rad51 recombinase...

  6. Regulation of hetDNA Length during Mitotic Double-Strand Break Repair in Yeast.

    Science.gov (United States)

    Guo, Xiaoge; Hum, Yee Fang; Lehner, Kevin; Jinks-Robertson, Sue

    2017-08-17

    Heteroduplex DNA (hetDNA) is a key molecular intermediate during the repair of mitotic double-strand breaks by homologous recombination, but its relationship to 5' end resection and/or 3' end extension is poorly understood. In the current study, we examined how perturbations in these processes affect the hetDNA profile associated with repair of a defined double-strand break (DSB) by the synthesis-dependent strand-annealing (SDSA) pathway. Loss of either the Exo1 or Sgs1 long-range resection pathway significantly shortened hetDNA, suggesting that these pathways normally collaborate during DSB repair. In addition, altering the processivity or proofreading activity of DNA polymerase δ shortened hetDNA length or reduced break-adjacent mismatch removal, respectively, demonstrating that this is the primary polymerase that extends both 3' ends. Data are most consistent with the extent of DNA synthesis from the invading end being the primary determinant of hetDNA length during SDSA. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The Role of Long Non Coding RNAs in the Repair of DNA Double Strand Breaks.

    Science.gov (United States)

    Dianatpour, Ali; Ghafouri-Fard, Soudeh

    2017-01-01

    DNA double strand breaks (DSBs) are abrasions caused in both strands of the DNA duplex following exposure to both exogenous and endogenous conditions. Such abrasions have deleterious effect in cells leading to genome rearrangements and cell death. A number of repair systems including homologous recombination (HR) and non-homologous end-joining (NHEJ) have been evolved to minimize the fatal effects of these lesions in cell. The role of protein coding genes in regulation of these pathways has been assessed previously. However, a number of recent studies have focused on evaluation of non-coding RNAs participation in DNA repair. We performed a computerized search of the Medline/ Pubmed databases with key words: DNA repair, homologous recombination, non-homologues end joining and long non-coding RNA (LncRNA). The existing data highlight the role of long non-coding RNAs in DSB repair as well as dysregulation in their expression which would lead to pathological conditions such as cancer. The specific mechanism of their contribution in DNA repair pathways has been elucidated for a few of them. LncRNAs participate in several steps of DNA repair pathways and regulate the expression of key components of these pathways including p53 tumor suppressor gene.

  8. Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Emad A. [Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Boer, Peter de [Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen (Netherlands); 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)

    2010-01-05

    The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of chromosome aberrations (CA) in the next generation. Since they are in a G1 comparable stage of the cell cycle, haploid spermatids are expected to repair DSBs by the non-homologous end-joining pathway (NHEJ). However, immunohistochemical evidence indicates that not all components of the classical NHEJ pathway are available since the presence of DNA-PKcs cannot be shown. Here, we demonstrate that round spermatids, as well as most other types of male germ cells express both Parp1 and XRCC1. Therefore, we have determined whether the alternative Parp1/XRCC1 dependent NHEJ pathway is active in these nuclei and also have tested for classical NHEJ activity by a genetic method. To evaluate DSB repair in SCID mice, deficient for DNA-PKcs, and to study the involvement of the Parp1/XRCC1 dependent NHEJ pathway in round spermatids, the loss of {gamma}-H2AX foci after irradiation has been determined in nucleus spreads of round spermatids of SCID mice and in nucleus spreads and histological sections of Parp1-inhibited mice and their respective controls. Results show that around half of the breaks in randomly selected round spermatids are repaired between 1 and 8 h after irradiation. The repair of 16% of the induced DSBs requires DNA-PKcs and 21% Parp1. Foci numbers in the Parp1-inhibited testes tend to be higher in spermatids of all epithelial stages reaching significance in stages I-III which indicates an active Parp1/XRCC1 pathway in round spermatids and a decreased repair capacity in later round spermatid stages. In Parp1-inhibited SCID mice only 14.5% of the breaks were repaired 8 h after irradiation indicating additivity of the two NHEJ pathways in round spermatids.

  9. RNF4 is required for DNA double-strand break repair in vivo

    DEFF Research Database (Denmark)

    Vyas, R; Kumar, R; Clermont, F

    2013-01-01

    , 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......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...... 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...

  10. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    DEFF Research Database (Denmark)

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance...... accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms....... In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity...

  11. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  12. 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)

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

    Science.gov (United States)

    Pontier, Daphne B; Tijsterman, Marcel

    2009-08-25

    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) or single-strand annealing (SSA). Here, we created a transgenic reporter system in C. elegans to investigate the relative contribution of these pathways in somatic cells during animal development. Although all three canonical pathways contribute to repair in the soma, in their combined absence, animals develop without growth delay and chromosomal breaks are still efficiently repaired. This residual repair, which we call alternative end-joining, dominates DSB repair only in the absence of NHEJ and resembles SSA, but acts independent of the SSA nuclease XPF and repair proteins from other pathways. The dynamic interplay between repair pathways might be developmentally regulated, because it was lost from terminally differentiated cells in adult animals. Our results demonstrate profound versatility in DSB repair pathways for somatic cells of C. elegans, which are thus extremely fit to deal with chromosomal breaks.

  14. The involvement of human RECQL4 in DNA double-strand break repair

    DEFF Research Database (Denmark)

    Singh, Dharmendra Kumar; Karmakar, Parimal; Aamann, Maria Diget

    2010-01-01

    Rothmund-Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas....... The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double-strand break (DSB) repair. The results show that RECQL4-deficient fibroblasts are moderately......-induced DSBs and remains for a shorter duration than WRN and BLM, indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with gammaH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N-terminus domain between amino...

  15. Assembly and function of DNA double-strand break repair foci in mammalian cells

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Mailand, Niels

    2010-01-01

    DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. One of the most discernible hallmarks...... of the cellular response to DSBs is the accumulation and local concentration of a plethora of DNA damage signaling and repair proteins in the vicinity of the lesion, initiated by ATM-mediated phosphorylation of H2AX (¿-H2AX) and culminating in the generation of distinct nuclear compartments, so-called Ionizing...... of such DNA repair foci still remains limited. In this review, we focus on recent discoveries on the mechanisms that govern the formation of IRIF, and discuss the implications of such findings in light of our understanding of the physiological importance of these structures....

  16. Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

    Energy Technology Data Exchange (ETDEWEB)

    Dynan, William S.; Li, Shuyi; Mernaugh, Raymond; Wragg, Stephanie; Takeda, Yoshihiko

    2003-03-27

    Exposure to low doses of ionizing radiation is universal. The signature injury from ionizing radiation exposure is induction of DNA double-strand breaks (DSBs). The first line of defense against DSBs is direct ligation of broken DNA ends via the nonhomologous end-joining pathway. Because even a relatively high environmental exposure induces only a few DSBs per cell, our current understanding of the response to this exposure is limited by the ability to measure DSB repair events reliably in situ at a single-molecule level. To address this need, we have taken advantage of biological amplification, measuring relocalization of proteins and detection of protein phosphorylation as a surrogate for detection of broken ends themselves. We describe the use of specific antibodies to investigate the kinetics and mechanism of repair of very small numbers of DSBs in human cells by the nonhomologous end-joining pathway.

  17. Epigenetic modifications in double-strand break DNA damage signaling and repair.

    Science.gov (United States)

    Rossetto, Dorine; Truman, Andrew W; Kron, Stephen J; Côté, Jacques

    2010-09-15

    Factors involved in the cellular response to double-strand break (DSB) DNA damage have been identified as potential therapeutic targets that would greatly sensitize cancer cells to radiotherapy and genotoxic chemotherapy. These targets could disable the repair machinery and/or reinstate normal cell-cycle checkpoint leading to growth arrest, senescence, and apoptosis. It is now clear that a major aspect of the DNA damage response occurs through specific interactions with chromatin structure and its modulation. It implicates highly dynamic posttranslational modifications of histones that are critical for DNA damage recognition and/or signaling, repair of the lesion, and release of cell-cycle arrest. Therefore, drugs that target the enzymes responsible for these modifications, or the protein modules reading them, have very high therapeutic potential. This review presents the current state of knowledge on the different chromatin modifications and their roles in each step of eukaryotic DSB DNA damage response. ©2010 AACR.

  18. 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...

  19. Double-strand break repair mechanisms in Escherichia coli: recent insights

    Directory of Open Access Journals (Sweden)

    Đermić D

    2015-01-01

    Full Text Available Damir Ðermić Ruđer Bošković Institute, Division of Molecular Biology, Zagreb, Croatia Abstract: In order to survive, all organisms must repair the continuous appearance of double-strand breaks (DSBs in their DNA. Escherichia coli does this by RecA-dependent homologous recombination (HR, during which the RecA protein is assembled on a 3´-terminated overhang that is created by a process called DNA end resection. The RecA nucleoprotein filament searches for and invades an intact homologous DNA sequence, creating a central HR intermediate. This review describes recent insights into HR and DSB repair in E. coli, especially the processes that precede the formation of a RecA nucleoprotein filament, with an emphasis on the regulation of 3´-tail metabolism. Since HR is a highly conserved process, the parallels to DSB repair in eukaryotic systems are discussed, bearing in mind that the lessons learned from studies in simpler bacterial models may be useful for studying DSB repair and the maintenance of genome stability in eukaryotes. Keywords: RecA nucleoprotein filament, homologous recombination, exonucleases, genome stability, 3´-overhang metabolism

  20. Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

    Directory of Open Access Journals (Sweden)

    Elise Darmon

    Full Text Available DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

  1. Repair of DNA strand breaks by the overlapping functions of lesion-specific and non-lesion-specific DNA 3' phosphatases.

    Science.gov (United States)

    Vance, J R; Wilson, T E

    2001-11-01

    In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3'-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3' phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3' processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal of 3' phosphates at strand breaks and does not possess more general 3' phosphodiesterase, exonuclease, or AP endonuclease activities. Deletion of TPP1 in an apn1 apn2 mutant background dramatically increased the sensitivity of the double mutant to DNA damage caused by H2O2 and bleomycin but not to damage caused by methyl methanesulfonate. The triple mutant was also deficient in the repair of 3' phosphate lesions left by Tdp1-mediated cleavage of camptothecin-stabilized Top1-DNA covalent complexes. Finally, the tpp1 apn1 apn2 triple mutation displayed synthetic lethality in combination with rad52, possibly implicating postreplication repair in the removal of unrepaired 3'-terminal lesions resulting from endogenous damage. Taken together, these results demonstrate a clear role for the lesion-specific enzyme, Tpp1, in the repair of a subset of DNA strand breaks.

  2. Repair of DNA Strand Breaks by the Overlapping Functions of Lesion-Specific and Non-Lesion-Specific DNA 3′ Phosphatases

    Science.gov (United States)

    Vance, John R.; Wilson, Thomas E.

    2001-01-01

    In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3′-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3′ phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3′ processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal of 3′ phosphates at strand breaks and does not possess more general 3′ phosphodiesterase, exonuclease, or AP endonuclease activities. Deletion of TPP1 in an apn1 apn2 mutant background dramatically increased the sensitivity of the double mutant to DNA damage caused by H2O2 and bleomycin but not to damage caused by methyl methanesulfonate. The triple mutant was also deficient in the repair of 3′ phosphate lesions left by Tdp1-mediated cleavage of camptothecin-stabilized Top1-DNA covalent complexes. Finally, the tpp1 apn1 apn2 triple mutation displayed synthetic lethality in combination with rad52, possibly implicating postreplication repair in the removal of unrepaired 3′-terminal lesions resulting from endogenous damage. Taken together, these results demonstrate a clear role for the lesion-specific enzyme, Tpp1, in the repair of a subset of DNA strand breaks. PMID:11585902

  3. Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Jessica M Moore

    2017-07-01

    Full Text Available Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB, II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR; damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS. First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage

  4. Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli.

    Science.gov (United States)

    Moore, Jessica M; Correa, Raul; Rosenberg, Susan M; Hastings, P J

    2017-07-01

    Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS

  5. Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair.

    Science.gov (United States)

    Mian, Elisabeth; Wiesmüller, Lisa

    2017-01-01

    Ataxia telangiectasia mutated (ATM) encodes a serine/threonine protein kinase, which is involved in various regulatory processes in mammalian cells. Its best-known role is apical activation of the DNA damage response following generation of DNA double-strand breaks (DSBs). When DSBs appear, sensor and mediator proteins are recruited, activating transducers such as ATM, which in turn relay a widespread signal to a multitude of downstream effectors. ATM mutation causes Ataxia telangiectasia (AT), whereby the disease phenotype shows differing characteristics depending on the underlying ATM mutation. However, all phenotypes share progressive neurodegeneration and marked predisposition to malignancies at the organismal level and sensitivity to ionizing radiation and chromosome aberrations at the cellular level. Expression and localization of the ATM protein can be determined via western blotting and immunofluorescence microscopy; however, detection of subtle alterations such as resulting from amino acid exchanges rather than truncating mutations requires functional testing. Previous studies on the role of ATM in DSB repair, which connects with radiosensitivity and chromosomal stability, gave at first sight contradictory results. To systematically explore the effects of clinically relevant ATM mutations on DSB repair, we engaged a series of lymphoblastoid cell lines (LCLs) derived from AT patients and controls. To examine DSB repair both in a quantitative and qualitative manners, we used an EGFP-based assay comprising different substrates for distinct DSB repair mechanisms. In this way, we demonstrated that particular signaling defects caused by individual ATM mutations led to specific DSB repair phenotypes. To explore the impact of ATM on carcinogenic chromosomal aberrations, we monitored chromosomal breakage at a breakpoint cluster region hotspot within the MLL gene that has been associated with therapy-related leukemia. PCR-based MLL-breakage analysis of HeLa cells

  6. 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

    2007-08-07

    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 gamma-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.

  7. Research on complex networks' repairing characteristics due to cascading failure

    Science.gov (United States)

    Chaoqi, Fu; Ying, Wang; Xiaoyang, Wang

    2017-09-01

    In reality, most of the topological structures of complex networks are not ideal. Considering the restrictions from all aspects, we cannot timely adjust and improve network defects. Once complex networks collapse under cascading failure, an appropriate repair strategy must be implemented. This repair process is divided into 3 kinds of situations. Based on different types of opening times, we presented 2 repair modes, and researched 4 kinds of repair strategies. Results showed that network efficiency recovered faster when the repair strategies were arranged in descending order by parameters under the immediate opening condition. However, the risk of secondary failure and additional expansion capacity were large. On the contrary, when repair strategies were in ascending order, the demand for additional capacity caused by secondary failure was greatly saved, but the recovery of network efficiency was relatively slow. Compared to immediate opening, delayed opening alleviated the contradiction between network efficiency and additional expansion capacity, particularly to reduce the risk of secondary failure. Therefore, different repair methods have different repair characteristics. This paper investigates the impact of cascading effects on the network repair process, and by presenting a detailed description of the status of each repaired node, helps us understand the advantages and disadvantages of different repair strategies.

  8. E4 ligase-specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis.

    Science.gov (United States)

    Ackermann, Leena; Schell, Michael; Pokrzywa, Wojciech; Kevei, Éva; Gartner, Anton; Schumacher, Björn; Hoppe, Thorsten

    2016-11-01

    Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome. In the absence of UFD-2, RAD-51 foci persist, and DNA damage-induced apoptosis is prevented. In contrast, UFD-2 foci are retained until recombination intermediates are removed by the Holliday-junction-processing enzymes GEN-1, MUS-81 or XPF-1. Formation of UFD-2 foci also requires proapoptotic CEP-1 (p53) signaling. Our findings establish a central role of UFD-2 in the coordination between the DNA-repair process and the apoptotic response.

  9. The non-homologous end-joining (NHEJ) pathway for the repair of DNA double-strand breaks: I. A mathematical model.

    Science.gov (United States)

    Taleei, Reza; Nikjoo, Hooshang

    2013-05-01

    This article presents a biochemical kinetic model for the non-homologous end joining (NHEJ) of DNA double-strand break (DSB) repair pathway. The model is part of a theoretical framework to encompass all cellular DSB repair pathways. The NHEJ model was developed by taking into consideration the biological characteristics of the repair processes in the absence of homologous recombination (HR), the major alternative pathway for DSB repair. The model considers fast and slow components of the repair kinetics resulting in a set of differential equations that were solved numerically. In the absence of available published data for reaction rate constants for the repair proteins involved in NHEJ, we propose reaction rate constants for the solution of the equations. We assume as a first approximation that the reaction rate constants are applicable to mammalian cells under same conditions. The model was tested by comparing measured and simulated DSB repair kinetics obtained with HR-deficient cell lines irradiated by X rays in the dose range of 20-80 Gy. Measured data for initial protein recruitment to a DSB were used to independently estimate rate constants for Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We show here based on the model of DSB repair described in this article, application of the model in the accompanying article (Taleei et al., Radiat. Res. 179, 540-548, 2013) and by simulation of repair times for each individual DSB produced by individual tracks of electrons, that the complexity of damage may explain the slow kinetics of DNA DSB repair.

  10. RecA bundles mediate homology pairing between distant sisters during DNA break repair

    Science.gov (United States)

    Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J.

    2014-02-01

    DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.

  11. End-joining repair of double-strand breaks in Drosophila melanogaster is largely DNA ligase IV independent.

    Science.gov (United States)

    McVey, Mitch; Radut, Dora; Sekelsky, Jeff J

    2004-12-01

    Repair of DNA double-strand breaks can occur by either nonhomologous end joining or homologous recombination. Most nonhomologous end joining requires a specialized ligase, DNA ligase IV (Lig4). In Drosophila melanogaster, double-strand breaks created by excision of a P element are usually repaired by a homologous recombination pathway called synthesis-dependent strand annealing (SDSA). SDSA requires strand invasion mediated by DmRad51, the product of the spn-A gene. In spn-A mutants, repair proceeds through a nonconservative pathway involving the annealing of microhomologies found within the 17-nt overhangs produced by P excision. We report here that end joining of P-element breaks in the absence of DmRad51 does not require Drosophila LIG4. In wild-type flies, SDSA is sometimes incomplete, and repair is finished by an end-joining pathway that also appears to be independent of LIG4. Loss of LIG4 does not increase sensitivity to ionizing radiation in late-stage larvae, but lig4 spn-A double mutants do show heightened sensitivity relative to spn-A single mutants. Together, our results suggest that a LIG4-independent end-joining pathway is responsible for the majority of double-strand break repair in the absence of homologous recombination in flies.

  12. Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing.

    Science.gov (United States)

    Soong, Chen-Pang; Breuer, Gregory A; Hannon, Ryan A; Kim, Savina D; Salem, Ahmed F; Wang, Guilin; Yu, Ruoxi; Carriero, Nicholas J; Bjornson, Robert; Sundaram, Ranjini K; Bindra, Ranjit S

    2015-02-01

    Efficient DNA double-strand break (DSB) repair is a critical determinant of cell survival in response to DNA damaging agents, and it plays a key role in the maintenance of genomic integrity. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. We now understand that HR and NHEJ repair are composed of multiple sub-pathways, some of which still remain poorly understood. As such, there is great interest in the development of novel assays to interrogate these key pathways, which could lead to the development of novel therapeutics, and a better understanding of how DSBs are repaired. Furthermore, assays which can measure repair specifically at endogenous chromosomal loci are of particular interest, because of an emerging understanding that chromatin interactions heavily influence DSB repair pathway choice. Here, we present the design and validation of a novel, next-generation sequencing-based approach to study DSB repair at chromosomal loci in cells. We demonstrate that NHEJ repair "fingerprints" can be identified using our assay, which are dependent on the status of key DSB repair proteins. In addition, we have validated that our system can be used to detect dynamic shifts in DSB repair activity in response to specific perturbations. This approach represents a unique alternative to many currently available DSB repair assays, which typical rely on the expression of reporter genes as an indirect read-out for repair. As such, we believe this tool will be useful for DNA repair researchers to study NHEJ repair in a high-throughput and sensitive manner, with the capacity to detect subtle changes in DSB repair patterns that was not possible previously. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Non-homologous end-joining protein expression screen from radiosensitive cancer patients yields a novel DNA double strand break repair phenotype.

    Science.gov (United States)

    McKay, Michael J; Goh, Su Kak; McKay, Jeremy N; Chao, Michael; McKay, Timothy M

    2017-03-01

    Clinical radiosensitivity is a significant impediment to tumour control and cure, in that it restricts the total doses which can safely be delivered to the whole radiotherapy population, within the tissue tolerance of potentially radiosensitive (RS) individuals. Understanding its causes could lead to personalization of radiotherapy. We screened tissues from a unique bank of RS cancer patients for expression defects in major DNA double-strand break repair proteins, using Western blot analysis and subsequently reverse-transcriptase polymerase chain reaction and pulsed-field gel electrophoresis. We hypothesized that abnormalities in expression of these proteins may explain the radiosensitivity of some of our cancer patients. The cells from one patient showed a reproducibly consistent expression reduction in two complex-forming DNA double-strand break repair protein components (DNA Ligase IV and XRCC4). We also showed a corresponding reduction in both gene products at the mRNA level. Additionally, the mRNA inducibility by ionizing radiation was increased for one of the proteins in the patient's cells. We confirmed the likely functional significance of the non-homologous end-joining (NHEJ) expression abnormalities with a DNA double strand break (DNA DSB) repair assay. We have identified a novel biological phenotype linked to clinical radiosensitivity. This is important in that very few molecular defects are known in human radiotherapy subjects. Such knowledge may contribute to the understanding of radiation response mechanisms in cancer patients and to personalization of radiotherapy.

  14. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

    Science.gov (United States)

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

  15. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

    Science.gov (United States)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

    2016-12-15

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts.

    Science.gov (United States)

    Solovjeva, Ljudmila; Firsanov, Denis; Vasilishina, Anastasia; Chagin, Vadim; Pleskach, Nadezhda; Kropotov, Andrey; Svetlova, Maria

    2015-10-12

    Studies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after several passages in standard conditions of cultivation due to what is known as "culture stress". Using proliferating young and non-dividing presenescent cells in primary cultures of Syrian hamster fibroblasts, we defined their response to the action of radiomimetic drug bleomycin (BL) that induces DNA double-strand breaks (DSBs). The effect of the drug was estimated by immunoblotting and immunofluorescence microscopy using the antibody to phosphorylated histone H2AX (gH2AX), which is generally accepted as a DSB marker. At all stages of the cell cycle, both presenescent and young cells demonstrated variability of the number of gH2AX foci per nucleus. gH2AX focus induction was found to be independent from BL-hydrolase expression. Some differences in DSB repair process between BL-treated young and presenescent Syrian hamster cells were observed: (1) the kinetics of gH2AX focus loss in G0 fibroblasts of young culture was faster than in cells that prematurely stopped dividing; (2) presenescent cells were characterized by a slower recruitment of DSB repair proteins 53BP1, phospho-DNA-PK and phospho-ATM to gH2AX focal sites, while the rate of phosphorylated ATM/ATR substrate accumulation was the same as that in young cells. Our results demonstrate an impairment of DSB repair in prematurely aged Syrian hamster fibroblasts in comparison with young fibroblasts, suggesting age-related differences in response to BL therapy.

  17. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

    Directory of Open Access Journals (Sweden)

    Michael Van Meter

    2016-09-01

    Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  18. Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse.

    NARCIS (Netherlands)

    M.J. McKay (Michael); C. Troelstra (Christine); P.J. van der Spek (Peter); R. Kanaar (Roland); B. Smit (Bep); A. Hagemeijer (Anne); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1996-01-01

    textabstractThe rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins

  19. DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks

    Directory of Open Access Journals (Sweden)

    Bray Clifford M

    2009-06-01

    Full Text Available Abstract Background DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability. Results Knockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs and also double strand breaks (DSBs, implicating AtLIG1 in repair of both these lesions. Conclusion Reduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.

  20. AUNIP/C1orf135 directs DNA double-strand breaks towards the homologous recombination repair pathway.

    Science.gov (United States)

    Lou, Jiangman; Chen, Hongxia; Han, Jinhua; He, Hanqing; Huen, Michael S Y; Feng, Xin-Hua; Liu, Ting; Huang, Jun

    2017-10-17

    DNA double-strand breaks (DSBs) are mainly repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). Here, we identify AUNIP/C1orf135, a largely uncharacterized protein, as a key determinant of DSB repair pathway choice. AUNIP physically interacts with CtIP and is required for efficient CtIP accumulation at DSBs. AUNIP possesses intrinsic DNA-binding ability with a strong preference for DNA substrates that mimic structures generated at stalled replication forks. This ability to bind DNA is necessary for the recruitment of AUNIP and its binding partner CtIP to DSBs, which in turn drives CtIP-dependent DNA-end resection and HR repair. Accordingly, loss of AUNIP or ablation of its ability to bind to DNA results in cell hypersensitivity toward a variety of DSB-inducing agents, particularly those that induce replication-associated DSBs. Our findings provide new insights into the molecular mechanism by which DSBs are recognized and channeled to the HR repair pathway.DNA double strand breaks can be repaired by homology-independent or homology-directed mechanisms. The choice between these pathways is a key event for genomic stability maintenance. Here the authors identify and characterize AUNIP, as a factor involved in tilting the balance towards homology repair.

  1. Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli.

    Science.gov (United States)

    Shee, Chandan; Gibson, Janet L; Darrow, Michele C; Gonzalez, Caleb; Rosenberg, Susan M

    2011-08-16

    Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example--a general stress-response-controlled switch to error-prone DNA break (double-strand break, DSB) repair--was suggested to be peculiar to an Escherichia coli F' conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F(-) E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation- and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS general-stress-response activator is expressed in unstressed cells, illustrating a stress-response-controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repair-dependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment.

  2. BRCA1 requirement for the fidelity of plasmid DNA double-strand break repair in cultured breast epithelial cells.

    Science.gov (United States)

    Thompson, Eric G; Fares, Hanna; Dixon, Kathleen

    2012-01-01

    The tumor suppressor breast cancer susceptibility protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks (DSBs). BRCA1 promotes the error-free repair of DSBs through homologous recombination and is also implicated in the regulation of nonhomologous end joining (NHEJ) repair fidelity. Here, we investigate the role of BRCA1 in NHEJ repair mutagenesis following a DSB. We examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of DSB repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of meiotic recombination enzyme 11 (Mre11) 3'-5'-exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA mutagenesis and MMEJ following a DSB. Inhibition of Mre11-exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid DSB repair. The results suggest that BRCA1 protects DNA from mutagenesis during nonhomologous DSB repair in plasmid-based assays. The increased frequency of DSB mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis. Copyright © 2011 Wiley Periodicals, Inc.

  3. Rad52 and Ku bind to different DNA structures produced early in double-strand break repair.

    Science.gov (United States)

    Ristic, Dejan; Modesti, Mauro; Kanaar, Roland; Wyman, Claire

    2003-09-15

    DNA double-strand breaks are repaired by one of two main pathways, non-homologous end joining or homologous recombination. A competition for binding to DNA ends by Ku and Rad52, proteins required for non-homologous end joining and homologous recombination, respectively, has been proposed to determine the choice of repair pathway. In order to test this idea directly, we compared Ku and human Rad52 binding to different DNA substrates. How ever, we found no evidence that these proteins would compete for binding to the same broken DNA ends. Ku bound preferentially to DNA with free ends. Under the same conditions, Rad52 did not bind preferentially to DNA ends. Using a series of defined substrates we showed that it is single-stranded DNA and not DNA ends that were preferentially bound by Rad52. In addition, Rad52 aggregated DNA, bringing different single-stranded DNAs in close proximity. This activity was independent of the presence of DNA ends and of the ability of the single-stranded sequences to form extensive base pairs. Based on these DNA binding characteristics it is unlikely that Rad52 and Ku compete as 'gatekeepers' of different DNA double-strand break repair pathways. Rather, they interact with different DNA substrates produced early in DNA double-strand break repair.

  4. Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4.

    Science.gov (United States)

    West, C E; Waterworth, W M; Jiang, Q; Bray, C M

    2000-10-01

    Rejoining of single- and double-strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double-strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by gamma-irradiation, represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP-dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino-acid sequence to the human DNA ligase IV. Yeast two-hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV-binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C-terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by gamma-irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.

  5. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation

    Science.gov (United States)

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-01-01

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation. PMID:25820262

  6. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation.

    Science.gov (United States)

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-05-12

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation. © 2015 The Authors.

  7. ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy.

    Science.gov (United States)

    Kato, Takamitsu A; Okayasu, Ryuichi; Jeggo, Penny A; Fujimori, Akira

    2011-12-01

    In a previous study using HiCEP (High coverage expression profiling), we demonstrated that ASPM (abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene was selectively down-regulated by IR (ionizing radiation). The roles of ASPM on radiosensitivity, however, have never been studied. Using glioblastoma cell lines and normal human fibroblasts, we investigated how IR sensitivity (survived fraction, DNA repair and chromosome aberration) was affected by the reduction of ASPM by specific siRNA (small interfering RNA). Down-regulation of ASPM by siRNA enhanced radiosensitivity in three human cell lines examined. Constant-field gel electrophoreses and γ-H2AX (phosphorylated form of Histone H2A variant H2AX) foci analysis showed that ASPM-specific siRNA impaired DNA double-strand breaks (DSB) in irradiated cells. Elevated levels of abnormal chromosomes were also observed following ASPM siRNA. In addition IR-sensitization by ASPM knockdown was not enhanced in DNA-PK (DNA-dependent protein kinase) deficient glioblastoma cells suggesting that ASPM impacts upon a DNA-PK-dependent pathway. Our results show for the first time that ASPM is required for efficient non-homologous end-joining in mammalian cells. In clinical applications, ASPM could be a novel target for combination therapy with radiation as well as a useful biomarker for tumor prognosis as ever described.

  8. An alternative pathway for Alu retrotransposition suggests a role in DNA double-strand break repair.

    Science.gov (United States)

    Srikanta, Deepa; Sen, Shurjo K; Huang, Charles T; Conlin, Erin M; Rhodes, Ryan M; Batzer, Mark A

    2009-03-01

    The Alu family is a highly successful group of non-LTR retrotransposons ubiquitously found in primate genomes. Similar to the L1 retrotransposon family, Alu elements integrate primarily through an endonuclease-dependent mechanism termed target site-primed reverse transcription (TPRT). Recent studies have suggested that, in addition to TPRT, L1 elements occasionally utilize an alternative endonuclease-independent pathway for genomic integration. To determine whether an analogous mechanism exists for Alu elements, we have analyzed three publicly available primate genomes (human, chimpanzee and rhesus macaque) for endonuclease-independent recently integrated or lineage specific Alu insertions. We recovered twenty-three examples of such insertions and show that these insertions are recognizably different from classical TPRT-mediated Alu element integration. We suggest a role for this process in DNA double-strand break repair and present evidence to suggest its association with intra-chromosomal translocations, in-vitro RNA recombination (IVRR), and synthesis-dependent strand annealing (SDSA).

  9. DNA Double Strand Break Repair and its Association with Inherited Predispositions to Breast Cancer

    Directory of Open Access Journals (Sweden)

    Scott Rodney J

    2004-02-01

    Full Text Available Abstract Mutations in BRCA1 account for the majority of familial aggregations of early onset breast and ovarian cancer (~70% and about 1/5 of all early onset breast cancer families; in contrast, mutations in BRCA2 account for a smaller proportion of breast/ovarian cancer families and a similar proportion of early onset breast cancer families. BRCA2 has also been shown to be associated with a much more pleiotropic disease spectrum compared to BRCA1. Since the identification of both BRCA1 and BRCA2 investigations into the functions of these genes have revealed that both are associated with the maintenance of genomic integrity via their apparent roles in cellular response to DNA damage, especially their involvement in the process of double strand DNA break repair. This review will focus on the specific roles of both genes and how functional differences may account for the diverse clinical findings observed between families that harbour BRCA1 or BRCA2 mutations.

  10. Crosstalk of DNA double-strand break repair pathways in PARP inhibitor treatment of BRCA1/2-mutated Cancer.

    Science.gov (United States)

    Sunada, Shigeaki; Nakanishi, Akira; Miki, Yoshio

    2018-02-10

    Germ-line mutations in breast cancer susceptibility gene 1 or 2 (BRCA1 or BRCA2) significantly increase cancer risk in hereditary breast and ovarian cancer syndrome (HBOC). Both genes function in the homologous recombination (HR) pathway of DNA double-strand break (DSB) repair process. Therefore, the DNA-repair defect characteristic in cancer cells brings therapeutic advantage for Poly(ADP-ribose) polymerase (PARP) inhibitor-induced synthetic lethality. The PARP inhibitor-based therapeutics initially causes cancer lethality but acquired resistance mechanisms have been found and need to be elucidated. In particular, it is essential to understand the mechanism of DNA damage and repair to PARP inhibitor treatment in detail. Further investigations have shown the roles of BRCA1/2 and its associations to other molecules in the DSB repair system. Notably, the repair pathway chosen in BRCA1-deficient cells could be entirely different from that in BRCA2-deficient cells after PARP inhibitor treatment. This review describes synthetic lethality and acquired resistance mechanisms to PARP inhibitor via the DSB repair pathway and subsequent repair process. In addition, recent knowledge of resistance mechanisms is discussed. Our model should contribute to the development of novel therapeutic strategies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  11. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene

    OpenAIRE

    Hollywood, Jennifer A.; Lee, Ciaran M.; Scallan, Martina F.; Harrison, Patrick T.

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 2...

  12. Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

    OpenAIRE

    Grzegorz Zapotoczny; Jeff Sekelsky

    2017-01-01

    DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess th...

  13. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    Science.gov (United States)

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

  14. Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.

    Science.gov (United States)

    Pennisi, Rosa; Antoccia, Antonio; Leone, Stefano; Ascenzi, Paolo; di Masi, Alessandra

    2017-08-01

    The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair. © 2017 Federation of European Biochemical Societies.

  15. Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms

    OpenAIRE

    Lambert, Sarah; Lopez, Bernard S.

    2000-01-01

    In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SceI cleavage site. We characterized conservative recombination after I-SceI cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two domi...

  16. DNA double-strand break repair: a theoretical framework and its application.

    Science.gov (United States)

    Murray, Philip J; Cornelissen, Bart; Vallis, Katherine A; Chapman, S Jon

    2016-01-01

    DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γH2AX. Many copies of γH2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti-γH2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo. Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, (111)In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti-γH2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti-γH2AX-TAT and γH2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti-γH2AX antibody is labelled with Auger electron-emitting (111)In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti-γH2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti-γH2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage

  17. More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny

    Directory of Open Access Journals (Sweden)

    Leyla Vahidi Ferdousi

    2014-11-01

    Full Text Available The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

  18. Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.

    Science.gov (United States)

    Zapotoczny, Grzegorz; Sekelsky, Jeff

    2017-04-03

    DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. Copyright © 2017 Zapotoczny and Sekelsky.

  19. Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    Grzegorz Zapotoczny

    2017-04-01

    Full Text Available DNA double-strand breaks (DSBs are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells.

  20. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    Science.gov (United States)

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  1. Mechanism of elimination of phosphorylated histone H2AX from chromatin after repair of DNA double-strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Svetlova, M.P., E-mail: svetlma@mail.ru [Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave., 194064 St. Petersburg (Russian Federation); Solovjeva, L.V.; Tomilin, N.V. [Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave., 194064 St. Petersburg (Russian Federation)

    2010-03-01

    Covalent modifications of histones in chromatin play an important role in regulation of eukaryotic gene expression and DNA repair. Formation of double-strand breaks (DSBs) in DNA is followed by the rapid local phosphorylation of the C-terminal serine in the replacement histone H2AX in megabase chromatin domains around DSBs and formation of discrete nuclear foci called {gamma}H2AX foci. This epigenetic modification of chromatin represents the 'histone code' for DNA damage signaling and repair and has been extensively studied during last decade. It is known that after DSB rejoining {gamma}H2AX foci are eliminated from the nucleus, but molecular mechanism of this elimination remains to be established. However, {gamma}H2AX elimination can serve as a useful marker of DSB repair in normal cells and tissues. In this paper the available data on kinetics and possible mechanisms of {gamma}H2AX elimination are reviewed.

  2. Laser-Guided Repair of Complex Bile Duct Strictures

    NARCIS (Netherlands)

    van Gulik, Thomas; Beek, Johan; de Reuver, Philip; Aronson, Daniel; van Delden, Otto; Busch, Olivier; Gouma, Dirk

    2009-01-01

    Background: The repair of bile duct strictures (BDS) requires identification of healthy bile duct proximal to the stenosis. Identification may be difficult in complex bile duct injuries after cholecystectomy or partial liver resection. Aim: We describe a technique to identify the prestenotic bile

  3. Laser-guided repair of complex bile duct strictures.

    NARCIS (Netherlands)

    Gulik, T. van; Beek, J.; Reuver, P. de; Aronson, D.C.; Delden, O. van; Busch, O.; Gouma, D.

    2009-01-01

    BACKGROUND: The repair of bile duct strictures (BDS) requires identification of healthy bile duct proximal to the stenosis. Identification may be difficult in complex bile duct injuries after cholecystectomy or partial liver resection. AIM: We describe a technique to identify the prestenotic bile

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

    Science.gov (United States)

    Shen, Hexi; Strunks, Gary D.; Klemann, Bart J. P. M.; Hooykaas, Paul J. J.; de Pater, Sylvia

    2016-01-01

    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. PMID:27866150

  5. 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.

  6. Apn1 and Apn2 endonucleases prevent accumulation of repair-associated DNA breaks in budding yeast as revealed by direct chromosomal analysis.

    Science.gov (United States)

    Ma, Wenjian; Resnick, Michael A; Gordenin, Dmitry A

    2008-04-01

    Base excision repair (BER) provides relief from many DNA lesions. While BER enzymes have been characterized biochemically, BER functions within cells are much less understood, in part because replication bypass and double-strand break (DSB) repair can also impact resistance to base damage. To investigate BER in vivo, we examined the repair of methyl methanesulfonate (MMS) induced DNA damage in haploid G1 yeast cells, so that replication bypass and recombinational DSB repair cannot occur. Based on the heat-lability of MMS-induced base damage, an assay was developed that monitors secondary breaks in full-length yeast chromosomes where closely spaced breaks yield DSBs that are observed by pulsed-field gel electrophoresis. The assay detects damaged bases and abasic (AP) sites as heat-dependent breaks as well as intermediate heat-independent breaks that arise during BER. Using a circular chromosome, lesion frequency and repair kinetics could be easily determined. Monitoring BER in single and multiple glycosylase and AP-endonuclease mutants confirmed that Mag1 is the major enzyme that removes MMS-damaged bases. This approach provided direct physical evidence that Apn1 and Apn2 not only repair cellular base damage but also prevent break accumulation that can result from AP sites being channeled into other BER pathway(s).

  7. Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Satoshi Nakajima

    Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

  8. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    Science.gov (United States)

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. Copyright © 2016. Published by Elsevier B.V.

  9. The Democratization of Gene Editing: Insights from site-specific cleavage and double-strand break repair

    Science.gov (United States)

    Jasin, Maria; Haber, James E.

    2017-01-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occur by homologous recombination that relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. PMID:27261202

  10. Approach to the classical radiation biology. Ionizing radiation effects and repair mechanism of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Utsumi, Hiroshi [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst

    2000-09-01

    Split-dose recovery has been observed under a variety of experimental conditions in many cell systems and believed to be the recovery of sublethal damage (SLD). It is considered to be one of the most widespread and important cellular responses in clinical radiotherapy. To study the molecular mechanism of this recovery, we analyzed the knockout mutants KU70{sup -/-}, RAD54{sup -/-}, and KU70{sup -/-}/ RAD54{sup -/-} of the chicken B-cell line, DT40. Rad54 participates in the homologous recombinational (HR) repair of DNA double-strand breaks (DSB), while Ku proteins are involved in non-homologous end-joining (NHEJ). Split-dose recovery was observed in the parent DT40 and KU70{sup -/-} cells. Moreover the split-dose survival enhancement had all of the characteristics of SLD recovery that had been demonstrated earlier: e.g., the reappearance of the shoulder of the survival curve with dose fractionation; repair at 25degC; and inhibition by the antibiotic actinomycin D. These results strongly suggest that SLD recovery is due to DSB repair via or mediated by HR, and that these breaks constitute SLD. The tonicity-sensitive potentially lethal damage (PLD) recovery was also found only in DT40 and KU70 {sup -/-} cells. Delayed-plating PLD recovery may be controlled by NHEJ repair that works through the cell cycle. These results lead to the conclusion that the repair of DSBs could explain the classical operational recovery phenomena. We have also investigated RBE/LET using those mutants. (author)

  11. Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

    Science.gov (United States)

    Odom, Obed W; Baek, Kwang-Hyun; Dani, Radhika N; Herrin, David L

    2008-03-01

    Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

  12. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Mirta M L Sousa

    Full Text Available Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs. Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ pathway of double-strand break (DSB repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel

  13. Mouse BAZ1A (ACF1 is dispensable for double-strand break repair but is essential for averting improper gene expression during spermatogenesis.

    Directory of Open Access Journals (Sweden)

    James A Dowdle

    2013-11-01

    Full Text Available ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations.

  14. Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-stranded break repair model for T-DNA integration.

    Science.gov (United States)

    Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

    2017-06-01

    Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-stranded break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-stranded break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration, which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosomes 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  15. The Caenorhabditis elegans WRN helicase promotes double-strand DNA break repair by mediating end resection and checkpoint activation.

    Science.gov (United States)

    Ryu, Jin-Sun; Koo, Hyeon-Sook

    2017-07-01

    The protein associated with Werner syndrome (WRN), is involved in DNA repair, checkpoint activation, and telomere maintenance. To better understand the involvement of WRN in double-strand DNA break (DSB) repair, we analyzed the combinatorial role of WRN-1, the Caenorhabditis elegans WRN helicase, in conjunction with EXO-1 and DNA-2 nucleases. We found that WRN-1 cooperates with DNA-2 to resect DSB ends in a pathway acting in parallel to EXO-1. The wrn-1 mutants show an aberrant accumulation of replication protein A (RPA) and RAD-51, and the same pattern of accumulation is also observed in checkpoint-defective strains. We conclude that WRN-1 plays a conserved role in the resection of DSB ends and mediates checkpoint signaling, thereby influencing levels of RPA and RAD-51. © 2017 Federation of European Biochemical Societies.

  16. Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time

    Science.gov (United States)

    Nakamura, Jun; Asakura, Shoji; Hester, Susan D.; de Murcia, Gilbert; Caldecott, Keith W.; Swenberg, James A.

    2003-01-01

    DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or during the base excision repair pathways. Here we established a new real-time assay to assess an imbalance of DNA SSB repair by indirectly measuring PARP-1 activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye. While this assay is not a direct method, it does not require DNA extraction or alkaline treatment, both of which could potentially cause an artifactual induction of SSBs. In addition, it takes only 4 h and requires less than a half million cells to perform this measurement. Using this assay, we demonstrated that the dose- and time-dependent depletion of NAD(P)H in XRCC1-deficient CHO cells exposed to methyl methanesulfonate. This decrease was almost completely blocked by a PARP inhibitor. Furthermore, methyl methanesulfonate reduced NAD(P)H in PARP-1+/+cells, whereas PARP-1–/– cells were more resistant to the decrease in NAD(P)H. These results indicate that the analysis of intracellular NAD(P)H level using water-soluble tetrazolium salt can assess an imbalance of SSB repair in living cells in real time. PMID:12930978

  17. Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes.

    Science.gov (United States)

    Andersen, Sabrina L; Sekelsky, Jeff

    2010-12-01

    Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers (COs) are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover (NCO) recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, it is becoming increasingly clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically-proliferating cells and that the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires COs) than mitotic recombination (repair of DNA damage, which typically generates NCOs). Copyright © 2010 WILEY Periodicals, Inc.

  18. Altered Hematopoiesis in Mice Lacking DNA Polymerase μ Is Due to Inefficient Double-Strand Break Repair

    Science.gov (United States)

    Lucas, Daniel; Escudero, Beatriz; Ligos, José Manuel; Segovia, Jose Carlos; Estrada, Juan Camilo; Terrados, Gloria; Blanco, Luis; Samper, Enrique; Bernad, Antonio

    2009-01-01

    Polymerase mu (Polμ) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polμ deficiency results in impaired Vκ-Jκ recombination and altered somatic hypermutation and centroblast development. In Polμ−/− mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body γ-irradiation revealed that Polμ also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polμ function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues. PMID:19229323

  19. Depletion of the bloom syndrome helicase stimulates homology-dependent repair at double-strand breaks in human chromosomes.

    Science.gov (United States)

    Wang, Yibin; Smith, Krissy; Waldman, Barbara Criscuolo; Waldman, Alan S

    2011-04-03

    Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.

    Science.gov (United States)

    Tadi, Satish Kumar; Sebastian, Robin; Dahal, Sumedha; Babu, Ravi K; Choudhary, Bibha; Raghavan, Sathees C

    2016-01-15

    Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders. © 2016 Tadi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  1. BLM–DNA2–RPA–MRN and EXO1–BLM–RPA–MRN constitute two DNA end resection machineries for human DNA break repair

    Science.gov (United States)

    Nimonkar, Amitabh V.; Genschel, Jochen; Kinoshita, Eri; Polaczek, Piotr; Campbell, Judith L.; Wyman, Claire; Modrich, Paul; Kowalczykowski, Stephen C.

    2011-01-01

    Repair of dsDNA breaks requires processing to produce 3′-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5′ → 3′ resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells. PMID:21325134

  2. Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB.

    Science.gov (United States)

    Badrinarayanan, Anjana; Le, Tung B K; Spille, Jan-Hendrik; Cisse, Ibrahim I; Laub, Michael T

    2017-05-01

    In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.

  3. The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair.

    Science.gov (United States)

    Kasten-Pisula, Ulla; Menegakis, Apostolos; Brammer, Ingo; Borgmann, Kerstin; Mansour, Wael Y; Degenhardt, Sarah; Krause, Mechthild; Schreiber, Andreas; Dahm-Daphi, Jochen; Petersen, Cordula; Dikomey, Ekkehard; Baumann, Michael

    2009-02-01

    Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms. Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and gammaH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay. Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD(50) for 10 fractions 46.0Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25Gy]) and cell cultures (D(0.01); 7.1 vs. 3.5Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells. The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.

  4. Double-strand break repair: are Rad51/RecA--DNA joints barriers to DNA replication?

    Science.gov (United States)

    Aguilera, A

    2001-06-01

    The central step of homologous recombination is the DNA strand exchange reaction catalyzed by bacterial RecA or eukaryotic Rad51. Besides Rad51-mediated synthesis-dependent strand annealing (SDSA), DNA ends can promote replication in Escherichia coli (recombination-dependent replication, RDR) and yeast (break-induced replication, BIR). However, what causes a DNA end to be repaired via SDSA or via BIR/RDR? I propose that Rad51/RecA--DNA plectonemic joints act as barriers to DNA replication and that BIR/RDR is only possible when the DNA polymerase that synthesizes DNA from the invading 3' end does not encounter RecA/Rad51--DNA joints in its path.

  5. SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint.

    Science.gov (United States)

    Carvalho, Sílvia; Vítor, Alexandra C; Sridhara, Sreerama C; Martins, Filipa B; Raposo, Ana C; Desterro, Joana M P; Ferreira, João; de Almeida, Sérgio F

    2014-05-06

    Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.DOI: http://dx.doi.org/10.7554/eLife.02482.001. Copyright © 2014, Carvalho et al.

  6. Tandem repeat modification during double-strand break repair induced by an engineered TAL effector nuclease in zebrafish genome.

    Directory of Open Access Journals (Sweden)

    Wanxu Huang

    Full Text Available Tandem repeats (TRs are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome in vivo. Transcription activator-like effector nucleases (TALENs are widely used in recent years in gene targeting for their specific binding to target sequences when engineered in vitro. Here, we show that the repair of a double-strand break (DSB induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.

  7. Midterm Follow-up after biventricular repair of the hypoplastic left heart complex

    NARCIS (Netherlands)

    Freund, JE; den Dekker, MH; Haas, F; Blank, AC; Freund, M.W.

    2015-01-01

    Background In neonates with hypoplastic left heart complex (HLHC), biventricular repair is considered superior to univentricular repair. The Z-scores of the mitral and the aortic valve annulus are primary factors for the choice of repair. Predictive cutoff values for the feasibility and optimal

  8. 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...

  9. Feasibility of measuring radiation-induced DNA double strand breaks and their repair by pulsed field gel electrophoresis in freshly isolated cells from the mouse RIF-1 tumor

    NARCIS (Netherlands)

    vanWaarde, MAWH; vanAssen, AJ; Konings, AWT; Kampinga, HH

    1996-01-01

    Purpose: To examine the technical feasibility of pulsed field gel electrophoresis (PFGE) as a predictive assay for the radioresponsiveness of tumors. Induction and repair of DNA double strand breaks (DSBs) in a freshly prepared cell suspension from a RIF-1 tumor (irradiated ex vivo) was compared

  10. Reconstitution and structure of a bacterial Pnkp1RnlHen1 RNA repair complex

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Pei; Selvadurai, Kiruthika; Huang , Raven H. (UIUC)

    2016-01-22

    Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. We report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins—Pnkp1, Rnl and Hen1—that are required to repair ribotoxin-cleaved RNA in vitro. The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1–Rnl–Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. Furthermore, the architecture and the locations of the active sites of the Pnkp1–Rnl–Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage.

  11. [Defect of preferential repair of gamma-ray-induced single-strand breaks in transcribed and non-transcribed DNA in Cockayne syndrome cells].

    Science.gov (United States)

    Igusheva, O A; Mikhel'son, V M; Pleskach, N M; Bil'din, V N; Zhestianikov, V D

    1998-01-01

    The repair of gamma-ray-induced DNA single-strand breaks in transcribed (protooncogene c-myc) and non-transcribed (human satellite III) DNA of normal human fibroblasts and fibroblasts obtained from a patient with Cockayne's syndrome (CS) has been investigated. A method of alkaline sucrose sedimentation was applied besides the Southern hybridization of 32P-DNA, containing sequences analysed with total 3H-DNA distributed through sucrose gradient fractions. No increase in the induction of DNA single-strand breaks was found in gamma-irradiated CS fibroblasts, compared to normal human fibroblasts. At the same time, an evident defect in the preferential repair of single-strand breaks in c-myc gene was observed.

  12. Symmetry-breaking polarization driven by a Cdc42p GEF-PAK complex.

    Science.gov (United States)

    Kozubowski, Lukasz; Saito, Koji; Johnson, Jayme M; Howell, Audrey S; Zyla, Trevin R; Lew, Daniel J

    2008-11-25

    In 1952, Alan Turing suggested that spatial patterns could arise from homogeneous starting conditions by feedback amplification of stochastic fluctuations. One example of such self-organization, called symmetry breaking, involves spontaneous cell polarization in the absence of spatial cues. The conserved GTPase Cdc42p is essential for both guided and spontaneous polarization, and in budding yeast cells Cdc42p concentrates at a single site (the presumptive bud site) at the cortex. Cdc42p concentrates at a random cortical site during symmetry breaking in a manner that requires the scaffold protein Bem1p. The mechanism whereby Bem1p promotes this polarization was unknown. Here we show that Bem1p promotes symmetry breaking by assembling a complex in which both a Cdc42p-directed guanine nucleotide exchange factor (GEF) and a Cdc42p effector p21-activated kinase (PAK) associate with Bem1p. Analysis of Bem1p mutants indicates that both GEF and PAK must bind to the same molecule of Bem1p, and a protein fusion linking the yeast GEF and PAK bypasses the need for Bem1p. Although mammalian cells lack a Bem1p ortholog, they contain more complex multidomain GEFs that in some cases can directly interact with PAKs, and we show that yeast containing an artificial GEF with similar architecture can break symmetry even without Bem1p. Yeast symmetry-breaking polarization involves a GEF-PAK complex that binds GTP-Cdc42p via the PAK and promotes local Cdc42p GTP-loading via the GEF. By generating fresh GTP-Cdc42p near pre-existing GTP-Cdc42p, the complex amplifies clusters of GTP-Cdc42p at the cortex. Our findings provide mechanistic insight into an evolutionarily conserved pattern-forming positive-feedback pathway.

  13. Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

    Directory of Open Access Journals (Sweden)

    Tamara Goldfarb

    2010-10-01

    Full Text Available Recombination between homologous chromosomes of different parental origin (homologs is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs] show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in

  14. Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast.

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    Arancha Sanchez

    2017-09-01

    Full Text Available The DNA repair enzyme polynucleotide kinase/phosphatase (PNKP protects genome integrity by restoring ligatable 5'-phosphate and 3'-hydroxyl termini at single-strand breaks (SSBs. In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (pnk1Δ cells of Schizosaccharomyces pombe. Here, we report that pnk1Δ cells have enhanced requirements for Rad3 (ATR/Mec1 and Chk1 checkpoint kinases, and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A (γH2A at damaged replication forks. The viability of pnk1Δ cells depends on Mre11 and Ctp1 (CtIP/Sae2 double-strand break (DSB resection proteins, Rad52 DNA strand annealing protein, Mus81-Eme1 Holliday junction resolvase, and Rqh1 (BLM/WRN/Sgs1 DNA helicase. Coupled with increased sister chromatid recombination and Rad52 repair foci in pnk1Δ cells, these findings indicate that lingering SSBs in pnk1Δ cells trigger Rad51-independent homology-directed repair of collapsed replication forks. From these data, we propose models for HDR-mediated tolerance of persistent SSBs with 3' phosphate in pnk1Δ cells.

  15. RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends.

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    Jim Westmoreland

    2009-09-01

    Full Text Available Resection of DNA double-strand break (DSB ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random "dirty-ended" DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE. We utilized this "PFGE-shift" to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after gamma-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1-2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.

  16. Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Noda, Taichi [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Kondo, Natsuko [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Mori, Eiichiro [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Okamoto, Noritomo [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nakagawa, Yosuke [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Ken [Department of Biology, Ibaraki Prefectual University of Health Sciences, 4669-2 Ami, Ami-mati, Inasiki-gun, Ibaraki 300-0394 (Japan); Zdzienicka, Malgorzata Z. [Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz (Poland); Thompson, Larry H. [Biosciences and Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808 (United States); Helleday, Thomas [Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ (United Kingdom); Department of Genetics, Microbiology and Toxicology Stockholm University, SE-106 91 Stockholm (Sweden); Asada, Hideo [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); and others

    2011-01-07

    The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA{sup -/-}, FANCC{sup -/-}, FANCA{sup -/-}C{sup -/-}, FANCD2{sup -/-} and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical {gamma}H2AX-staining assay. Although the sensitivity of FANCA{sup -/-}, FANCC{sup -/-} and FANCA{sup -/-}C{sup -/-} cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2{sup -/-} cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, {gamma}H2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex. -- Research highlights: {yields} We examined to clarify the repair pathways of formaldehyde-induced DNA damage. Formaldehyde induces DNA double strand breaks (DSBs). {yields} DSBs are repaired through the Fanconi anemia (FA) repair pathway. {yields} This pathway is independent of the FA nuclear core complex. {yields} We also found that homologous recombination repair was induced by formaldehyde.

  17. Molecular Process Producing Oncogene Fusion in Lung Cancer Cells by Illegitimate Repair of DNA Double-Strand Breaks

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    Yoshitaka Seki

    2015-09-01

    Full Text Available Constitutive activation of oncogenes by fusion to partner genes, caused by chromosome translocation and inversion, is a critical genetic event driving lung carcinogenesis. Fusions of the tyrosine kinase genes ALK (anaplastic lymphoma kinase, ROS1 (c-ros oncogene 1, or RET (rearranged during transfection occur in 1%–5% of lung adenocarcinomas (LADCs and their products constitute therapeutic targets for kinase inhibitory drugs. Interestingly, ALK, RET, and ROS1 fusions occur preferentially in LADCs of never- and light-smokers, suggesting that the molecular mechanisms that cause these rearrangements are smoking-independent. In this study, using previously reported next generation LADC genome sequencing data of the breakpoint junction structures of chromosome rearrangements that cause oncogenic fusions in human cancer cells, we employed the structures of breakpoint junctions of ALK, RET, and ROS1 fusions in 41 LADC cases as “traces” to deduce the molecular processes of chromosome rearrangements caused by DNA double-strand breaks (DSBs and illegitimate joining. We found that gene fusion was produced by illegitimate repair of DSBs at unspecified sites in genomic regions of a few kb through DNA synthesis-dependent or -independent end-joining pathways, according to DSB type. This information will assist in the understanding of how oncogene fusions are generated and which etiological factors trigger them.

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

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    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.

  19. The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

    DEFF Research Database (Denmark)

    Torres-Rosell, Jordi; Sunjevaric, Ivana; De Piccoli, Giacomo

    2007-01-01

    Homologous recombination (HR) is crucial for maintaining genome integrity by repairing DNA double-strand breaks (DSBs) and rescuing collapsed replication forks. In contrast, uncontrolled HR can lead to chromosome translocations, loss of heterozygosity, and deletion of repetitive sequences. Contro...

  20. Scimitar Syndrome-Complex Surgical Revision 3 Decades After Repair.

    Science.gov (United States)

    Abadeer, Maher N; Stuth, Eckehard A E; Kouretas, Peter C; Ginde, Salil; Jacobsen, Roni; Woods, Ronald K

    2017-02-01

    We present a case of a 39-year-old woman with scimitar syndrome who had a 2-patch repair 3 decades previously and presented with a right-to-left shunt of the inferior vena cava (IVC) to the left atrium resulting from baffle dehiscence. We discuss details of our reoperative repair. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  1. A novel protein, Rsf1/Pxd1, is critical for the single-strand annealing pathway of double-strand break repair in Schizosaccharomyces pombe.

    Science.gov (United States)

    Wang, Hanqian; Zhang, Zhanlu; Zhang, Lan; Zhang, Qiuxue; Zhang, Liang; Zhao, Yangmin; Wang, Weibu; Fan, Yunliu; Wang, Lei

    2015-06-01

    The process of single-strand annealing (SSA) repairs DNA double-strand breaks that are flanked by direct repeat sequences through the coordinated actions of a series of proteins implicated in recombination, mismatch repair and nucleotide excision repair (NER). Many of the molecular and mechanistic insights gained in SSA repair have principally come from studies in the budding yeast Saccharomyces cerevisiae. However, there is little molecular understanding of the SSA pathway in the fission yeast Schizosaccharomyces pombe. To further our understanding of this important process, we established a new chromosome-based SSA assay in fission yeast. Our genetic analyses showed that, although many homologous components participate in SSA repair in these species indicating that some evolutionary conservation, Saw1 and Slx4 are not principal agents in the SSA repair pathway in fission yeast. This is in marked contrast to the function of Saw1 and Slx4 in budding yeast. Additionally, a novel genus-specific protein, Rsf1/Pxd1, physically interacts with Rad16, Swi10 and Saw1 in vitro and in vivo. We find that Rsf1/Pxd1 is not required for NER and demonstrate that, in fission yeast, Rsf1/Pxd1, but not Saw1, plays a critical role in SSA recombination. © 2015 John Wiley & Sons Ltd.

  2. COMPLEX SIMULATION MODEL OF TRAIN BREAKING-UP PROCESS AT THE HUMPS

    Directory of Open Access Journals (Sweden)

    E. B. Demchenko

    2015-11-01

    Full Text Available Purpose. One of the priorities of station sorting complex functioning improvement is the breaking-up process energy consumptions reduction, namely: fuel consumption for train pushing and electric energy consumption for cut braking. In this regard, an effective solution of the problem of energy consumption reduction at breaking-up subsystem requires a comprehensive handling of train pushing and cut rolling down processes. At the same time, the analysis showed that the current task of pushing process improvement and cut rolling down effectiveness increase are solved separately. To solve this problem it is necessary to develop the complex simulation model of train breaking up process at humps. Methodology. Pushing process simulation was done based on adapted under the shunting conditions traction calculations. In addition, the features of shunting locomotives work at the humps were taken into account. In order to realize the current pushing mode the special algorithm of hump locomotive controlling, which along with the safety shunting operation requirements takes into account behavioral factors associated with engineer control actions was applied. This algorithm provides train smooth acceleration and further movement with speed, which is close to the set speed. Hump locomotive fuel consumptions were determined based on the amount of mechanical work performed by locomotive traction. Findings. The simulation model of train pushing process was developed and combined with existing cut rolling down model. Cut initial velocity is determined during simulation process. The obtained initial velocity is used for further cut rolling process modeling. In addition, the modeling resulted in sufficiently accurate determination of the fuel rates consumed for train breaking-up. Originality. The simulation model of train breaking-up process at the humps, which in contrast to the existing models allows reproducing complexly all the elements of this process in detail

  3. In what extent classic radiation biology can be understand at the molecular level? Biological effects of ionizing radiation and repair mechanism of double strand breaks of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Utsumi, Hiroshi [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst.

    2000-09-01

    This review revealed that classic radiation biology phenomena not based on substantial concept, such as lethal damage, sublethal damage, potentially lethal damage, recoveries from them, variation of radiation sensitivity due to cell cycle, RBE/LET relationships and mathematical expression of survival curve, can be described only at the levels of DNA double strand breaks (DSB) and its repair mechanism. Authors investigated chicken cells with DSB repair-defect, analyzed the molecular mechanisms of 'Elkind recovery' where cell survival rates are higher after fractionated irradiation than after single irradiation at a fixed dose, and showed that the phenomenon was derived from the cell revival resulting from homologous recombination repair of DSB yielded on homologous chromosome DNA. Investigations using HIMAC also revealed the molecular mechanisms of RBE/LET in those cells. Findings indicate that biological effects of ionizing radiation can be described at the molecular level.(K.H.)

  4. Influence of Double-Strand Break Repair on Radiation Therapy-Induced Acute Skin Reactions in Breast Cancer Patients

    Energy Technology Data Exchange (ETDEWEB)

    Mumbrekar, Kamalesh Dattaram [Division of Radiobiology and Toxicology, School of Life Sciences, Manipal University, Manipal, Karnataka (India); Fernandes, Donald Jerard [Department of Radiotherapy and Oncology, Shirdi Sai Baba Cancer Hospital and Research Centre, Kasturba Hospital, Manipal, Karnataka (India); Goutham, Hassan Venkatesh [Division of Radiobiology and Toxicology, School of Life Sciences, Manipal University, Manipal, Karnataka (India); Sharan, Krishna [Department of Radiotherapy and Oncology, Shirdi Sai Baba Cancer Hospital and Research Centre, Kasturba Hospital, Manipal, Karnataka (India); Vadhiraja, Bejadi Manjunath [Manipal Hospital, Bangalore, Karnataka (India); Satyamoorthy, Kapaettu [Division of Biotechnology, School of Life Sciences, Manipal University, Manipal, Karnataka (India); Bola Sadashiva, Satish Rao, E-mail: satishraomlsc@gmail.com [Division of Radiobiology and Toxicology, School of Life Sciences, Manipal University, Manipal, Karnataka (India)

    2014-03-01

    Purpose: Curative radiation therapy (RT)-induced toxicity poses strong limitations for efficient RT and worsens the quality of life. The parameter that explains when and to what extent normal tissue toxicity in RT evolves would be of clinical relevance because of its predictive value and may provide an opportunity for personalized treatment approach. Methods and Materials: DNA double-strand breaks and repair were analyzed by microscopic γ-H2AX foci analysis in peripheral lymphocytes from 38 healthy donors and 80 breast cancer patients before RT, a 2 Gy challenge dose of x-ray exposed in vitro. Results: The actual damage (AD) at 0.25, 3, and 6 hours and percentage residual damage (PRD) at 3 and 6 hours were used as parameters to measure cellular radiosensitivity and correlated with RT-induced acute skin reactions in patients stratified as non-overresponders (NOR) (Radiation Therapy Oncology Group [RTOG] grade <2) and overresponders (OR) (RTOG grade ≥2). The results indicated that the basal and induced (at 0.25 and 3 hours) γ-H2AX foci numbers were nonsignificant (P>.05) between healthy control donors and the NOR and OR groups, whereas it was significant between ORs and healthy donors at 6 hours (P<.001). There was a significantly higher PRD in OR versus NOR (P<.05), OR versus healthy donors (P<.001) and NOR versus healthy donors (P<.01), supported further by the trend analysis (r=.2392; P=.0326 at 6 hours). Conclusions: Our findings strongly suggest that the measurement of PRD by performing γ-H2AX foci analysis has the potential to be developed into a clinically useful predictive assay.

  5. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

    Science.gov (United States)

    Deniz, Miriam; Kaufmann, Julia; Stahl, Andreea; Gundelach, Theresa; Janni, Wolfgang; Hoffmann, Isabell; Keimling, Marlen; Hampp, Stephanie; Ihle, Michaela; Wiesmüller, Lisa

    2016-11-01

    Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis. © FASEB.

  6. Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines.

    Science.gov (United States)

    Bourton, Emma C; Plowman, Piers N; Zahir, Sheba Adam; Senguloglu, Gonul Ulus; Serrai, Hiba; Bottley, Graham; Parris, Christopher N

    2012-02-01

    The measurement of γ-H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ-H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5-SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA-PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a (60)Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5-SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24-hr period was due to the failure in the repair of DNA DSB. However, in the DNA-PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through-put analysis of foci induction in a variety of cell types. Copyright © 2011 International Society for Advancement of Cytometry.

  7. Annealing of Complementary DNA Sequences During Double-Strand Break Repair in Drosophila Is Mediated by the Ortholog of SMARCAL1.

    Science.gov (United States)

    Holsclaw, Julie Korda; Sekelsky, Jeff

    2017-05-01

    DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. We hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. We used unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP-binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP-binding defect mutant showed reduced end joining, shedding light on the interaction between SDSA and end-joining pathways. Copyright © 2017 by the Genetics Society of America.

  8. 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.

  9. Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

    Science.gov (United States)

    Cesare, Anthony J

    2014-11-01

    Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

  10. mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster.

    Science.gov (United States)

    Portin, Petter

    2005-12-01

    The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.

  11. The Anterior Preperitoneal Approach for Repair of Complex Inguinal Hernias

    Directory of Open Access Journals (Sweden)

    Safa Onel

    2014-08-01

    Results: A total of 40 patients (Male:32, Female:8 underwent hernia repair with our technique during the study period. The mean age was (+/-SD 44+/-6.8 years. Four patients had giant direct hernia,12 patients had giant inguino-scrotal hernia, 14 patients had recurrent and 10 patients had femoral hernia. 4 patients were underwent emergent surgery due to incarceration. The mean operation time was (+/-SD 61+/-11 min. There was no recurrence with the mean follow-up time of 7+/-2.2 years. Cnclusion: Our surgical technique allows to repair all types of inguinal hernia with one piece of prolene mesh by covering all potential defects. [Cukurova Med J 2014; 39(4.000: 822-828

  12. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

    NARCIS (Netherlands)

    Vriend, Lianne E. M.; Prakash, Rohit; Chen, Chun-Chin; Vanoli, Fabio; Cavallo, Francesca; Zhang, Yu; Jasin, Maria; Krawczyk, Przemek M.

    2016-01-01

    DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided

  13. Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

    DEFF Research Database (Denmark)

    Liberti, Sascha E; Andersen, Sofie Dabros; Wang, Jing

    2011-01-01

    Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phas...

  14. Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme - onset of the enzymatic repair process

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1999-12-01

    To describe the first step of the enzymatic repair process (formation of complex enzyme-DNA), in which the thymine dimer (TD) part is removed from DNA, the 500 picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center - Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation the research was focused on the role of electrostatic energy in formation of complex enzyme-DNA. It is found, that during the first 100 ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Walls interactions with TD part of DNA and forms complex that lasts stabile for 500 ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD ({approx} +10 kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stabile. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT ({approx} -11 kcal/mol), in contrary to the positive one in the system with TD, doesn't drive enzyme towards DNA and complex is not formed. (author)

  15. Repair of DNA Strand Breaks by the Overlapping Functions of Lesion-Specific and Non-Lesion-Specific DNA 3′ Phosphatases

    OpenAIRE

    Vance, John R.; Wilson, Thomas E.

    2001-01-01

    In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3′-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3′ phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3′ processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal o...

  16. ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

    Science.gov (United States)

    Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M; Bianco, Piero R; Surtees, Jennifer A

    2014-06-01

    In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Paul F.; Nham, Peter B.; Urbin, Salustra S.; Hinz, John M.; Jones, Irene M. [Biosciences and Biotechnology Division, PO Box 808, L-452, Lawrence Livermore National Laboratory, Livermore, CA, 94551-0808 (United States); Thompson, Larry H., E-mail: thompson14@llnl.gov [Biosciences and Biotechnology Division, PO Box 808, L-452, Lawrence Livermore National Laboratory, Livermore, CA, 94551-0808 (United States)

    2010-01-05

    DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of {sup 137}Cs {gamma}-rays. Quiescent G{sub 0}/G{sub 1}-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24 h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV {approx} 0.2) than the level of spontaneous foci, which ranged from 0.2-2.6 foci/cell (CV {approx} 0.6; mean {+-} SD of 1.00 {+-} 0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24 h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24 h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.

  18. ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by Mismatch and Double-strand Break Repair DNA substrates

    Science.gov (United States)

    Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M.; Bianco, Piero R.; Surtees, Jennifer A.

    2014-01-01

    In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. PMID:24746922

  19. Bose-Einstein condensation and symmetry breaking of a complex charged scalar field

    Energy Technology Data Exchange (ETDEWEB)

    Matos, Tonatiuh [Centro de Investigacion y de Estudios Avanzados del IPN, Departamento de Fisica, Mexico, DF (Mexico); Castellanos, Elias [Centro de Investigacion y de Estudios Avanzados del IPN, Departamento de Fisica, Mexico, DF (Mexico); Universidad Autonoma de Chiapas, Mesoamerican Centre for Theoretical Physics, Tuxtla Gutierrez, Chiapas (Mexico); Suarez, Abril [Centro de Investigacion y de Estudios Avanzados del IPN, Departamento de Fisica, Mexico, DF (Mexico); Universidad Politecnica Metropolitana de Hidalgo, Departamento de Aeronautica, Tolcayuca, Hidalgo (Mexico)

    2017-08-15

    In this work the Klein-Gordon equation for a complex scalar field with U(1) symmetry endowed in a mexican-hat scalar field potential with thermal and electromagnetic contributions is written as a Gross-Pitaevskii (GP)-like equation. This equation is interpreted as a charged generalization of the GP equation at finite temperatures found in previous works. Its hydrodynamical representation is obtained and the corresponding thermodynamical properties are derived and related to measurable quantities. The condensation temperature in the non-relativistic regime associated with the aforementioned system within the semiclassical approximation is calculated. Also, a generalized equation for the conservation of energy for a charged bosonic gas is found when electromagnetic fields are introduced, and it is studied how under certain circumstances its breaking of symmetry can give some insight on the phase transition of the system not just into the condensed phase but also on other related systems. (orig.)

  20. Single-molecule fluorescence microscopy on nucleotide excision repair complexes using GFP fusion proteins

    NARCIS (Netherlands)

    Segers-Nolten, Gezina M.J.; Rademakers, Suzanne; Vermeulen, Wim; Lenferink, Aufrid T.M.; Otto, Cornelis; Hoeijmakers, Jan; Greve, Jan; Koenig, Karsten; Tanke, Hans J.; Schneckenburger, Herbert

    2000-01-01

    Scanning Confocal Fluorescence Microscopy is used for single molecule studies on DNA-protein complexes that occur in Nucleotide Excision Repair (NER). During DNA-damage elimination by the NER-pathway, complex protein structures assemble over DNA. It is our aim to resolve the architecture of these

  1. Robotic Mitral Valve Repair for Simple and Complex Degenerative Disease: Midterm Clinical and Echocardiographic Quality Outcomes.

    Science.gov (United States)

    Suri, Rakesh M; Taggarse, Amit; Burkhart, Harold M; Daly, Richard C; Mauermann, William; Nishimura, Rick A; Li, Zhuo; Dearani, Joseph A; Michelena, Hector I; Enriquez-Sarano, Maurice

    2015-11-24

    Severe primary (degenerative) mitral regurgitation (MR) is repaired with durable results when simple single-scallop disease is addressed. The midterm quality outcomes of minimally invasive repair for complex disease are unknown, however. From January 2008 to January 2015, 487 patients (56±11 years, 360 men, ejection fraction 65±6%, 98.8% complete follow-up) underwent robotic mitral valve repair for severe nonischemic degenerative MR. Simple pathology was addressed in 289 of 487 (59%) patients, and complex repair (all others) was performed in 198 of 487 (41%). Four patients died during follow-up with a 5-year survival rate 99.5% (99.4% simple; 99.5% complex; hazard ratio, 0.48; 95% confidence interval, 0.05-4.59); and New York Heart Association functional class I/II was documented in 97.9% (477/487). Eight patients had recurrence of moderate-to-severe MR (4 simple, 4 complex), with a 5-year freedom from MR of 94.6% (96.2% simple; 92.7%, complex; P=0.67; hazard ratio, 1.36; 95% confidence interval, 0.34-5.43). Seven patients (2 simple, 5 complex), underwent mitral reoperation, with a 5-year freedom from reoperation of 97.7% (99.1% simple; 95.7% complex; P=0.13; hazard ratio, 3.35; 95% confidence interval, 0.65-17.32). At a large tertiary care referral center, midterm quality outcomes after robotic correction of degenerative MR are excellent, with very high survival, infrequent complications, and a low likelihood of MR recurrence, regardless of mitral valve repair complexity. Awareness of these improvements in outcome is important to inform contemporary decisions regarding high-quality alternatives to conventional and percutaneous mitral repair. © 2015 American Heart Association, Inc.

  2. Kidney repair and stem cells: a complex and controversial process.

    Science.gov (United States)

    Yeagy, Brian A; Cherqui, Stephanie

    2011-09-01

    Over the last decade, stem cells have been the topic of much debate and investigation for their regenerative potential in the case of renal injury. This review focuses on bone marrow stem cells (BMSC) for renal repair and the potential origins of the controversial results between studies. Some authors have shown that BMSC can differentiate into renal cells and reverse renal dysfunction while others obtained contradictory results. One significant variation between these studies is the choice of BMSC used. According to the literature and our own experience, unfractionated bone marrow cells and hematopoietic stem cells are able to lead to long-term cell tissue engraftment and repair, whereas mesenchymal stem cells have a short-term paracrine effect. Detection of the bone-marrow-derived cells is also an important source of error. However, the major difference between studies is the model of kidney injury used. Two categories of models have to be distinguished: acute and chronic kidney disease. However, variation within these categories also exists. The outcomes of various strategies for BMSC transplantation after injury to the kidney must be compared within a single model and cannot be transposed from one model to another.

  3. The Association of Low-Penetrance Variants in DNA Repair Genes with Colorectal Cancer: A Systematic Review and Meta-Analysis

    OpenAIRE

    Aggarwal, Nikhil; Donald, Neil D; Malik, Salim; Selvendran, Subothini S; McPhail, Mark JW.; Monahan, Kevin J

    2017-01-01

    Objectives: Approximately 35% of colorectal cancer (CRC) risk is attributable to heritable factors known hereditary syndromes, accounting for 6%. The remainder may be due to lower penetrance polymorphisms particularly of DNA repair genes. DNA repair pathways, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), direct reversal repair (DRR), and double-strand break repair are complex, evolutionarily conserved, and critical in carcinogenesis. Germline m...

  4. Identification of Proteins Required for Repair of Double-Strand Chromosome Breaks, a Predisposing Factor in Breast Cancer

    National Research Council Canada - National Science Library

    Marshall-Batty, Kimberly

    2001-01-01

    ... when replication forks become arrested. This project bas focused on developing a bacterial model for DSB repair by characterizing the enzymatic apparatus needed to initiate DNA replication on recombination intermediates...

  5. Identification of Proteins Required for Repair of Double-Strand Chromosome Breaks, a Predisposing Factor in Breast Cancer

    National Research Council Canada - National Science Library

    Jones, Jessica

    2000-01-01

    ... when replication forks become arrested. This project has focused on developing a bacterial model for DSB repair by characterizing the enzymatic apparatus needed to initiate DNA replication on recombination intermediates...

  6. 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.

  7. MCM8 is required for a pathway of meiotic double-strand break repair independent of DMC1 in Arabidopsis thaliana.

    Science.gov (United States)

    Crismani, Wayne; Portemer, Virginie; Froger, Nicole; Chelysheva, Liudmila; Horlow, Christine; Vrielynck, Nathalie; Mercier, Raphaël

    2013-01-01

    Mini-chromosome maintenance (MCM) 2-9 proteins are related helicases. The first six, MCM2-7, are essential for DNA replication in all eukaryotes. In contrast, MCM8 is not always conserved in eukaryotes but is present in Arabidopsis thaliana. MCM8 is required for 95% of meiotic crossovers (COs) in Drosophila and is essential for meiosis completion in mouse, prompting us to study this gene in Arabidopsis meiosis. Three allelic Atmcm8 mutants showed a limited level of chromosome fragmentation at meiosis. This defect was dependent on programmed meiotic double-strand break (DSB) formation, revealing a role for AtMCM8 in meiotic DSB repair. In contrast, CO formation was not affected, as shown both genetically and cytologically. The Atmcm8 DSB repair defect was greatly amplified in the absence of the DMC1 recombinase or in mutants affected in DMC1 dynamics (sds, asy1). The Atmcm8 fragmentation defect was also amplified in plants heterozygous for a mutation in either recombinase, DMC1 or RAD51. Finally, in the context of absence of homologous chromosomes (i.e. haploid), mutation of AtMCM8 also provoked a low level of chromosome fragmentation. This fragmentation was amplified by the absence of DMC1 showing that both MCM8 and DMC1 can promote repair on the sister chromatid in Arabidopsis haploids. Altogether, this establishes a role for AtMCM8 in meiotic DSB repair, in parallel to DMC1. We propose that MCM8 is involved with RAD51 in a backup pathway that repairs meiotic DSB without giving CO when the major pathway, which relies on DMC1, fails.

  8. MCM8 is required for a pathway of meiotic double-strand break repair independent of DMC1 in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Wayne Crismani

    Full Text Available Mini-chromosome maintenance (MCM 2-9 proteins are related helicases. The first six, MCM2-7, are essential for DNA replication in all eukaryotes. In contrast, MCM8 is not always conserved in eukaryotes but is present in Arabidopsis thaliana. MCM8 is required for 95% of meiotic crossovers (COs in Drosophila and is essential for meiosis completion in mouse, prompting us to study this gene in Arabidopsis meiosis. Three allelic Atmcm8 mutants showed a limited level of chromosome fragmentation at meiosis. This defect was dependent on programmed meiotic double-strand break (DSB formation, revealing a role for AtMCM8 in meiotic DSB repair. In contrast, CO formation was not affected, as shown both genetically and cytologically. The Atmcm8 DSB repair defect was greatly amplified in the absence of the DMC1 recombinase or in mutants affected in DMC1 dynamics (sds, asy1. The Atmcm8 fragmentation defect was also amplified in plants heterozygous for a mutation in either recombinase, DMC1 or RAD51. Finally, in the context of absence of homologous chromosomes (i.e. haploid, mutation of AtMCM8 also provoked a low level of chromosome fragmentation. This fragmentation was amplified by the absence of DMC1 showing that both MCM8 and DMC1 can promote repair on the sister chromatid in Arabidopsis haploids. Altogether, this establishes a role for AtMCM8 in meiotic DSB repair, in parallel to DMC1. We propose that MCM8 is involved with RAD51 in a backup pathway that repairs meiotic DSB without giving CO when the major pathway, which relies on DMC1, fails.

  9. LncRNA lnc-RI regulates homologous recombination repair of DNA double-strand breaks by stabilizing RAD51 mRNA as a competitive endogenous RNA.

    Science.gov (United States)

    Shen, Liping; Wang, Qi; Liu, Ruixue; Chen, Zhongmin; Zhang, Xueqing; Zhou, Pingkun; Wang, Zhidong

    2017-12-04

    DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. The current models of the mechanism of DSB repair are based on studies of DNA repair proteins. Long non-coding RNAs (lncRNAs) have recently emerged as new regulatory molecules, with diverse functions in biological processes. In the present study, we found that expression of the ionizing radiation-inducible lncRNA, lnc-RI, was correlate negatively with micronucleus frequencies in human peripheral blood lymphocytes. Knockdown of lnc-RI significantly increased spontaneous DSBs levels, which was confirmed to be associated with the decreased efficiency of homologous recombination (HR) repair of DSBs. The expression of RAD51, a key recombinase in the HR pathway, decreased sharply in lnc-RI-depressed cells. In a further investigation, we demonstrated that miR-193a-3p could bind with both lnc-RI and RAD51 mRNA and depressed the expression of lnc-RI and RAD51 mRNA. Lnc-RI acted as a competitive endogenous RNA (ceRNA) to stabilize RAD51 mRNA via competitive binding with miR-193a-3p and release of its inhibition of RAD51 expression. To our knowledge, this is the first study to demonstrate the role of lnc-RI in regulating HR repair of DSBs. The feedback loop established in the current study suggests that lnc-RI is critical for the maintenance of genomic stability. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Genetic variants in DNA double-strand break repair genes and risk of salivary gland carcinoma: a case-control study.

    Directory of Open Access Journals (Sweden)

    Li Xu

    Full Text Available DNA double strand break (DSB repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC. We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs and 95% confidence intervals (CIs. Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs for the variant alleles of these SNPs were 1.71 (1.40-2.09, P = 1.70 × 10(-7 and 0.58 (0.45-0.74, P = 2.00 × 10(-5 respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR = 2.21, 95% CI: 1.55-3.15, P = 1.25 × 10(-5, n = 74, and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR = 0.60, 95% CI: 0.42-0.87, P = 6.91 × 10(-3, n = 123. Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings.

  11. Live Cell Imaging to Study Real-Time ATM-Mediated Recruitment of DNA Repair Complexes to Sites of Ionizing Radiation-Induced DNA Damage.

    Science.gov (United States)

    Jakob, Burkhard; Taucher-Scholz, Gisela

    2017-01-01

    Measurements of protein recruitment and the formation of repair complexes at DNA double-strand breaks in real time provide valuable insight into the regulation of the early DNA damage response. Here, we describe the use of live cell microscopy in combination with ionizing radiation as a tool to evaluate the influence of ATM and its site-specific phosphorylation of target proteins on these processes. Recommendations are made for the preparation of the cells and the design of specialized cell chambers for the localized (and/or targeted) irradiation with charged particles at accelerator beamlines as well as the microscopic equipment and protocol to obtain high-resolution, sensitive fluorescence measurements.

  12. On the break down of reality at superluminal velocities, Quantum entanglement and Singularities (Complex Universe)

    Science.gov (United States)

    Estakhr, Ahmad Reza

    2017-09-01

    In the real world nothing can move faster than the speed of light. But what convinces you that our world is all real? I realized that reality break down at superluminal velocities (By studying the physics of tachyonic neutrinos), Quantum entanglement and Singularities of Black Holes, I realized that infact our world is complex and has two parts, one part of the world is real (the part that nothing can move faster than the speed of light) but the other part of the world is imaginary. z = a + ib Einstein was wrong because he thought our world is completely real (Of course he was not alone in this belief almost all physicists believe that our world is completely real) Eventually his false interpretation of reality censored imaginary part of the universe. Einstein's Second Postulate of special theory of relativity was a misleading guide to the true nature of reality. He `expected' the true nature of reality will follow to his (false) postulate, But the true nature of reality is unlike what anyone ever `expected'!. Einstein twist facts to suit his theory of relativity instead of theories to suit facts!. This is a dramatic revisions to our conception of the theory of relativity, Reality is complex but We always perceive its real part.

  13. Regulation of 53BP1 protein stability by RNF8 and RNF168 is important for efficient DNA double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Yiheng Hu

    Full Text Available 53BP1 regulates DNA double-strand break (DSB repair. In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ pathway, and this activity was dependent upon RNF8 and RNF168. We observed that 53BP1 protein was diffusely abundant in nuclei, and upon ionizing radiation, 53BP1 was everywhere degraded except at DNA damage sites. Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF did not form. Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished. Our data suggest a novel mechanism for responding to DSB that upon ionizing radiation, 53BP1 was divided into two populations, ensuring functional DSB repair: damage site-bound 53BP1 whose binding signal is known to be generated by RNF8 and RNF168; and unbound bulk 53BP1 whose ensuing degradation is regulated by RNF8 and RNF168.

  14. Endogenous sequence patterns predispose the repair modes of CRISPR/Cas9-induced DNA double-stranded breaks in Arabidopsis thaliana.

    Science.gov (United States)

    Vu, Giang T H; Cao, Hieu X; Fauser, Friedrich; Reiss, Bernd; Puchta, Holger; Schubert, Ingo

    2017-10-01

    The possibility to predict the outcome of targeted DNA double-stranded break (DSB) repair would be desirable for genome editing. Furthermore the consequences of mis-repair of potentially cell-lethal DSBs and the underlying pathways are not yet fully understood. Here we study the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced mutation spectra at three selected endogenous loci in Arabidopsis thaliana by deep sequencing of long amplicon libraries. Notably, we found sequence-dependent genomic features that affected the DNA repair outcome. Deletions of 1-bp to 1 kbp (all due to NHEJ) and deletions combined with insertions between 5-bp to >100 bp [caused by a synthesis-dependent strand annealing (SDSA)-like mechanism] occurred most frequently at all three loci. The appearance of single-stranded annealing events depends on the presence and distance between repeats flanking the DSB. The frequency and size of insertions is increased if a sequence with high similarity to the target site was available in cis. Most deletions were linked to pre-existing microhomology. Deletion and/or insertion mutations were blunt-end ligated or via de novo generated microhomology. While most mutation types and, to some degree, their predictability are comparable with animal systems, the broad range of deletion mutations seems to be a peculiar feature of the plant A. thaliana. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  15. Dynamics of MutS-mismatched DNA complexes are predictive of their repair phenotypes.

    Science.gov (United States)

    DeRocco, Vanessa C; Sass, Lauryn E; Qiu, Ruoyi; Weninger, Keith R; Erie, Dorothy A

    2014-04-01

    MutS recognizes base-base mismatches and base insertions/deletions (IDLs) in newly replicated DNA. Specific interactions between MutS and these errors trigger a cascade of protein-protein interactions that ultimately lead to their repair. The inability to explain why different DNA errors are repaired with widely varying efficiencies in vivo remains an outstanding example of our limited knowledge of this process. Here, we present single-molecule Förster resonance energy transfer measurements of the DNA bending dynamics induced by Thermus aquaticus MutS and the E41A mutant of MutS, which is known to have error specific deficiencies in signaling repair. We compared three DNA mismatches/IDLs (T-bulge, GT, and CC) with repair efficiencies ranging from high to low. We identify three dominant DNA bending states [slightly bent/unbent (U), intermediately bent (I), and significantly bent (B)] and find that the kinetics of interconverting among states varies widely for different complexes. The increased stability of MutS-mismatch/IDL complexes is associated with stabilization of U and lowering of the B to U transition barrier. Destabilization of U is always accompanied by a destabilization of B, supporting the suggestion that B is a "required" precursor to U. Comparison of MutS and MutS-E41A dynamics on GT and the T-bulge suggests that hydrogen bonding to MutS facilitates the changes in base-base hydrogen bonding that are required to achieve the U state, which has been implicated in repair signaling. Taken together with repair propensities, our data suggest that the bending kinetics of MutS-mismatched DNA complexes may control the entry into functional pathways for downstream signaling of repair.

  16. Resurfacing the Penis of Complex Hypospadias Repair ("Hypospadias Cripples").

    Science.gov (United States)

    Fam, Mina M; Hanna, Moneer K

    2017-03-01

    After the creation of a neourethra in a "hypospadias cripple," resurfacing the penis with healthy skin is a significant challenge because local tissue is often scarred and unusable. We reviewed our experience with various strategies to resurface the penis of hypospadias cripples. We retrospectively reviewed the records of 215 patients referred after multiple unsuccessful hypospadias repairs from 1981 to 2014. In 130 of 215 patients we performed resurfacing using local penile flaps using various techniques, including Byars flaps, Z-plasty or double Z-plasty, or a dorsal relaxing incision. Of the 215 patients 85 did not have adequate healthy local penile skin to resurface the penis after urethroplasty. Scrotal skin was used to resurface the penis in 54 patients, 6 underwent tissue expansion of the dorsal penile skin during a 12 to 16-week period prior to penile resurfacing, 23 underwent full-thickness skin grafting and another 4 received a split-thickness skin graft. Of the 56 patients who underwent fasciomyocutaneous rotational flaps, tissue expansion or a combination of both approaches 54 (96.4%) finally had a successful outcome. All 6 patients who underwent tissue expansion had a successful outcome without complications and were reported on previously. All 23 full-thickness skin grafts took with excellent results. All 4 patients who underwent fenestrated split-thickness skin grafting had 100% graft take but secondary contraction and ulceration were associated with sexual activity. In our experience scrotal skin flaps, tissue expansion of the dorsal penile skin and full-thickness skin grafts serve as reliable approaches in resurfacing the penis in almost any hypospadias cripple lacking healthy local skin. Copyright © 2017 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  17. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

    Energy Technology Data Exchange (ETDEWEB)

    Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy; Schild, David; Wiese, Claudia

    2010-06-29

    To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

  18. Use of rigid internal fixation in the repair of complex and comminuted mandible fractures.

    Science.gov (United States)

    Klotch, D

    1987-08-01

    An overview of the use of rigid internal fixation using the AO method for the repair of complex mandibular injuries is presented. The end result of such an approach is the return of the patient as rapidly as possible to his or her normal status with return to normal alimentation, normal mobility of the mandible, normal airway, and normal access to the oral cavity.

  19. Complex catalysts from self-repairing ensembles to highly reactive air-based oxidation systems

    Science.gov (United States)

    Craig L. Hill; Laurent Delannoy; Dean C. Duncan; Ira A. Weinstock; Roman F. Renneke; Richard S. Reiner; Rajai H. Atalla; Jong Woo Han; Daniel A. Hillesheim; Rui Cao; Travis M. Anderson; Nelya M. Okun; Djamaladdin G. Musaev; Yurii V. Geletii

    2007-01-01

    Progress in four interrelated catalysis research efforts in our laboratory are summarized: (1) catalytic photochemical functionalization of unactivated CeH bonds by polyoxometalates (POMs); (2) self-repairing catalysts; (3) catalysts for air-based oxidations under ambient conditions; and (4) terminal oxo complexes of the late-transition metal elements and their...

  20. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes.

    Directory of Open Access Journals (Sweden)

    Sandrine Lacoste

    Full Text Available Patients who undergo autologous hematopoietic stem cell transplantation (aHCT for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML. Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC, which is thought to influence the effect chemotherapeutic treatments have on the patient's stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA] are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER or nucleotide excision repair (NER. We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year. To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average, aHCT patients (both cases and controls showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was

  1. A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans.

    Directory of Open Access Journals (Sweden)

    Esma Bentchikou

    2010-01-01

    Full Text Available In Deinococcus radiodurans, the extreme resistance to DNA-shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a DeltarecA mutant: DeltarecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to gamma-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, DeltauvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of DeltauvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.

  2. A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

    Science.gov (United States)

    Bentchikou, Esma; Servant, Pascale; Coste, Geneviève; Sommer, Suzanne

    2010-01-01

    In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA. PMID:20090937

  3. The sandwich technique for repair of pectus carinatum and excavatum/carinatum complex.

    Science.gov (United States)

    Park, Hyung Joo; Kim, Kyung Soo

    2016-09-01

    Simple external compression of pectus carinatum seems to have its limitations, particularly the repair of asymmetric pectus carinatum or excavatum/carinatum complex. We devised the sandwich technique (press-molding) to remodel the entire chest wall. The purpose of this study is to introduce the sandwich technique and appraise the early results. Between January 2007 and January 2016, 523 consecutive patients with pectus carinatum and its variants were analyzed retrospectively. No patients were excluded during the study period. The sandwich 1 and 2 techniques using the internal and external pectus bars were for pectus carinatum repair. Modified techniques using the external string and the internal bar were to treat the lower costal flare (the flare-buster) and focal protuberances (the magic string) in pectus excavatum repair. Statistical analyses were carried out using paired and unpaired t-test or Wilcoxon signed rank tests. The sandwich repair with the external and internal bars was applied to 58 pectus carinatum patients: seven symmetric (12.1%), 14 asymmetric (24.1%), and 37 carinatum-excavatum complex (63.8%). After pectus excavatum repair, 426 patients had the flare-buster and 39 patients received the magic string. The sandwich 1 technique achieved near-complete resolution of carinatum in 52 patients (86.2%). The sandwich 2 technique accomplished almost symmetric configuration with no residual carinatum in all six cases. The sandwich technique using the external and internal bars seems to be effective in treating asymmetric pectus carinatum and complex excavatum/carinatum deformities. The flare-buster and the magic string effectively relieve the costal flare and focal protuberances in pectus excavatum repair.

  4. DNA excision repair and double-strand break repair gene polymorphisms and the level of chromosome aberration in children with long-term exposure to radon.

    Science.gov (United States)

    Larionov, Aleksey V; Sinitsky, Maxim Y; Druzhinin, Vladimir G; Volobaev, Valentin P; Minina, Varvara I; Asanov, Maxim A; Meyer, Alina V; Tolochko, Tatiana A; Kalyuzhnaya, Ekaterina E

    2016-08-01

    To study polymorphic variants of repair genes in people affected by long-term exposure to radon. The chromosome aberration frequency in peripheral blood lymphocytes was used as the biological marker of genotoxicity. Genotyping of 12 single nucleotide polymorphisms in DNA repair genes (APE, XRCC1, OGG1, ADPRT, XpC, XpD, XpG, Lig4 and NBS1) was performed in children with long-term resident exposure to radon. Quantification of the aberrations was performed using light microscopy. The total frequency of aberrations was increased in carriers of the G/G genotype for the XpD gene (rs13181) polymorphism in recessive model confirmed by the results of ROC-analysis ('satisfactory predictor', AUC = 0.609). Single chromosome fragments frequency was increased in carriers of the G/G genotype in comparison with the T/T genotype. In respect to the total frequency of aberrations, the G/G genotype for the XpG gene (rs17655) polymorphism was also identified as a 'satisfactory predictor' (AUC = 0.605). Carriers of the T/C genotype for the ADPRT gene (rs1136410) polymorphism were characterized by an increased level of single fragments relative to the T/T genotype. The relationships with several types of cytogenetic damage suggest these three SNP (rs13181, rs17655 and rs1136410) may be considered radiosensitivity markers.

  5. Retinal detachment with a break at pars plicata associated with congenital malformation of the lens-zonule-ciliary body complex.

    Science.gov (United States)

    Tsai, Fang-Yi; Lau, Ling-Ing; Chen, Shih-Jen; Lee, Fenq-Lih

    2015-01-01

    Retinal detachment with a break at the pars plicata associated with congenital malformation of lens-zonule-ciliary body complex is rare; most reports are of young Japanese male patients with atopic dermatitis. The present case report is the first to describe the condition in a Chinese patient with no atopic dermatitis or trauma history. A 22-year-old male presented with blurred vision in the left eye for 4 months. Fundus examination revealed shallow lower temporal retinal detachment. Further examination with scleral indentation under maximal pupil dilatation identified a break at the far periphery beyond the ora serrata and pars plana. Gonioscopy revealed a pars plicata break at the nonpigmented ciliary epithelium associated with congenital ciliary process hypoplasia and subtle lens defect at the same meridian. The retina was successfully reattached after segmental scleral buckling, cryopexy, and laser photocoagulation.

  6. Recruitment Kinetics of DNA Repair Proteins Mdc1 and Rad52 but Not 53BP1 Depend on Damage Complexity

    Science.gov (United States)

    Hable, Volker; Drexler, Guido A.; Brüning, Tino; Burgdorf, Christian; Greubel, Christoph; Derer, Anja; Seel, Judith; Strickfaden, Hilmar; Cremer, Thomas; Friedl, Anna A.; Dollinger, Günther

    2012-01-01

    The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET  = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T0 after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ1. Mdc1 accumulates faster (T0 = 17±2 s, τ1 = 98±11 s) than 53BP1 (T0 = 77±7 s, τ1 = 310±60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T0 = 73±16 s, τ1 = 1050±270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ1 of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies. PMID:22860035

  7. Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but not 53BP1 depend on damage complexity.

    Directory of Open Access Journals (Sweden)

    Volker Hable

    Full Text Available The recruitment kinetics of double-strand break (DSB signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/µm to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T(0 after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ(1. Mdc1 accumulates faster (T(0 = 17 ± 2 s, τ(1 = 98 ± 11 s than 53BP1 (T(0 = 77 ± 7 s, τ(1 = 310 ± 60 s after high LET irradiation. However, recruitment of Mdc1 slows down (T(0 = 73 ± 16 s, τ(1 = 1050 ± 270 s after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ(1 of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.

  8. A staged repair of crossed fused renal ectopia with complex abdominal aortic and iliac aneurysms

    Directory of Open Access Journals (Sweden)

    Mary Teresa O'Donnell, MD

    2016-09-01

    Full Text Available Crossed fused renal ectopia in the presence of abdominal aortic aneurysms (AAAs is a rare challenge to therapy. A 68-year-old man had a 6.9-cm AAA, a 6.6-cm left common iliac aneurysm, and a 3.2-cm right internal iliac aneurysm. He had multiple comorbidities and a right-sided crossed fused kidney supplied by two small renal arteries arising from the AAA. Aortorenal bypass with staged endovascular aneurysm repair was used to treat the aneurysmal disease. Two-stage open aortorenal bypass followed by endovascular repair for complex aneurysms is feasible in the rare patient with such a congenital anomaly.

  9. Two stage hybrid approach for complex aortic coarctation repair

    Directory of Open Access Journals (Sweden)

    Crockett James

    2009-02-01

    Full Text Available Abstract Background Management of an adult patient with aortic coarctation and an associated cardiac pathology poses a great surgical challenge since there are no standard guidelines for the therapy of such complex pathology. Debate exists not only on which lesion should be corrected first, but also upon the type and timing of the procedure. Surgery can be one- or two-staged. Both of these strategies are accomplice with elevate morbidity and mortality. Case report In the face of such an extended surgical approach, balloon dilatation seems preferable for treatment of severe aortic coarctation. We present an adult male patient with aortic coarctation combined with ascending aorta aneurysm and concomitant aortic valve regurgitation. The aortic coarctation was corrected first, using percutaneous balloon dilatation; and in a second stage the aortic regurgitation and ascending aorta aneurysm was treated by Bentall procedure. The patients' postoperative period was uneventful. Three years after the operation he continues to do well.

  10. Elevated presence of retrotransposons at sites of DNA double strand break repair in mouse models of metabolic oxidative stress and MYC-induced lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Rockwood, Lynne D.; Felix, Klaus; Janz, Siegfried

    2004-04-14

    The chromosomally integrated shuttle vector pUR288 contains a lacZ reporter gene to study mutagenesis in vivo. We used pUR288 to compare patterns of genomic instability in two mouse models, lymphoma resulting from deregulated c-MYC expression ({lambda}-MYC), and endogenous oxidative stress caused by partial glucose 6-phosphate dehydrogenase (G6PD) deficiency. We found previously that spontaneous mutations in both models were predominantly genomic rearrangements of lacZ with mouse sequences, while most mutations in controls were point mutations. Here, we characterized the fine structure of 68 lacZ/mouse rearrangements from {lambda}-MYC lymphomas and G6PD deficient mice by sequencing breakpoint junctions and determining the origin of recombining mouse sequences. Fifty-eight of 68 (85%) recombination partners were identified. The structure of rearrangements from both {lambda}-MYC and G6PD deficient mice were remarkably alike. Intra-chromosomal deletions and inversions were common, occurring in 41% (24/58) of rearrangements, while 59% (34/58) were random translocations between lacZ and other chromosomes. Signatures of double strand break repair by nonhomologous end joining were observed at breakpoint junctions; 37% (25/68) contained 1-4 bp microhomologies, while the remaining breakpoints had no sequence homology. Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons, which constitute {approx}10% of the mouse genome, were present at 25% (17/68) of breakpoints, suggesting their participation in rearrangements. The similarity in the structure of rearrangements is consistent with the hypothesis that genetic rearrangements in {lambda}-MYC lymphomas and G6PD deficient mice result from the same mechanism, mutagenic repair of DNA double strand breaks arising from oxidative damage.

  11. 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

  12. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    Directory of Open Access Journals (Sweden)

    Anita Collavoli

    2011-01-01

    Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  13. Combined Laparoscopic and Perineal Approach to Omental Interposition Repair of Complex Rectovaginal Fistula.

    Science.gov (United States)

    de Bruijn, Hester; Maeda, Yasuko; Murphy, Jamie; Warusavitarne, Janindra; Vaizey, Carolynne J

    2018-01-01

    Surgical repair of rectovaginal fistula remains a challenge. Complex and recurrent rectovaginal fistula repairs often fail because of scarring and devascularization of the surrounding tissue. Omental interposition may promote healing by introducing bulky vascularized tissue into the rectovaginal septum. With the patient in the lithotomy position, the rectovaginal septum was dissected transperineally up to the fistula tract and the openings on both vaginal and rectal sides were closed using interrupted, absorbable sutures. The dissection was continued cranially to meet the laparoscopic dissection from above. The laparoscopic surgeon detached the omentum from the colon, then the anastomotic arterial branches between the Barlow's arcade and the gastroepiploic arcade were divided and the greater omentum was mobilized, retaining blood supply from the left gastroepiploic artery. The rectum was then mobilized commencing on the right lateral side of the mesorectum and then proceeding anteriorly. The peritoneum between the rectum and the vagina was incised and the anterior mobilization was continued to connect with the perineal dissection. The mobilized omentum was pulled down between the rectum and the vagina.The perineal operator secured the omentum around the rectal closure and at skin level with absorbable sutures. All of the patients had a defunctioning ileostomy or colostomy before omental repair. Patients underwent repair for complex or recurrent rectovaginal fistulas with this novel approach. Fistula healing was evaluated during examination under anaesthesia. All of the patients had completely healed at the latest follow-up (median = 15 mo; range, 8-41 mo). Postoperative complications included 1 superficial wound infection that was treated conservatively and 1 rectovaginal hematoma, which required CT-guided aspiration. Combined laparoscopic omental interposition with perineal rectovaginal fistula repair is a safe and effective treatment for complex rectovaginal

  14. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast.

    Science.gov (United States)

    Ira, Grzegorz; Malkova, Anna; Liberi, Giordano; Foiani, Marco; Haber, James E

    2003-11-14

    Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.

  15. Srs2 and Sgs1–Top3 Suppress Crossovers during Double-Strand Break Repair in Yeast

    Science.gov (United States)

    Ira, Grzegorz; Malkova, Anna; Liberi, Giordano; Foiani, Marco; Haber, James E.

    2015-01-01

    Summary Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Δ cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange. PMID:14622595

  16. DNA breaks and repair in interstitial telomere sequences: Influence of chromatin structure; Etude des cassures de l'ADN et des mecanismes de reparation dans les sequences telomeriques interstitielles: Influence de la structure chromatinienne

    Energy Technology Data Exchange (ETDEWEB)

    Revaud, D.

    2009-06-15

    Interstitial Telomeric Sequences (ITS) are over-involved in spontaneous and radiationinduced chromosome aberrations in chinese hamster cells. We have performed a study to investigate the origin of their instability, spontaneously or after low doses irradiation. Our results demonstrate that ITS have a particular chromatin structure: short nucleotide repeat length, less compaction of the 30 nm chromatin fiber, presence of G-quadruplex structures. These features would modulate breaks production and would favour the recruitment of alternative DNA repair mechanisms, which are prone to produce chromosome aberrations. These pathways could be at the origin of chromosome aberrations in ITS whereas NHEJ and HR Double Strand Break repair pathways are rather required for a correct repair in these regions. (author)

  17. Break in the Heat Capacity Change at 303 K for Complex Binding of Netropsin to AATT Containing Hairpin DNA Constructs

    Science.gov (United States)

    Freyer, Matthew W.; Buscaglia, Robert; Hollingsworth, Amy; Ramos, Joseph; Blynn, Meredith; Pratt, Rachael; Wilson, W. David; Lewis, Edwin A.

    2007-01-01

    Studies performed in our laboratory demonstrated the formation of two thermodynamically distinct complexes on binding of netropsin to a number of hairpin-forming DNA sequences containing AATT-binding regions. These two complexes were proposed to differ only by a bridging water molecule between the drug and the DNA in the lower affinity complex. A temperature-dependent isothermal titration calorimetry (ITC)-binding study was performed using one of these constructs (a 20-mer hairpin of sequence 5′-CGAATTCGTCTCCGAATTCG) and netropsin. This study demonstrated a break in the heat capacity change for the formation of the complex containing the bridging water molecule at ∼303 K. In the plot of the binding enthalpy change versus temperature, the slope (ΔCp) was −0.67 kcal mol−1 K−1 steeper after the break at 303 K. Because of the relatively low melting temperature of the 20-mer hairpin (341 K (68°C)), the enthalpy change for complex formation might have included some energy of refolding of the partially denatured hairpin, giving the suggestion of a larger ΔCp. Studies done on the binding of netropsin to similar constructs, a 24-mer and a 28-mer, with added GC basepairs in the hairpin stem to increase thermal stability, exhibit the same nonlinearity in ΔCp over the temperature range of from 275 to 333 K. The slopes (ΔCp) were −0.69 and −0.64 kcal mol−1 K−1 steeper after 303 K for the 24-mer and 28-mer, respectively. This observation strengthens the argument regarding the presence of a bridging water molecule in the lower affinity netropsin/DNA complex. The ΔCp data seem to infer that because the break in the heat capacity change function for the lower affinity binding occurs at the isoequilibrium temperature for water, water may be included or trapped in the complex. The fact that this break does not occur in the heat capacity change function for formation of the higher affinity complex can similarly be taken as evidence that water is not included in

  18. The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function.

    Science.gov (United States)

    Xue, Lian; Furusawa, Yoshiya; Okayasu, Ryuichi; Miura, Masahiko; Cui, Xing; Liu, Cuihua; Hirayama, Ryoichi; Matsumoto, Yoshitaka; Yajima, Hirohiko; Yu, Dong

    2015-01-01

    DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

    Science.gov (United States)

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-09-01

    5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2.

    Science.gov (United States)

    Tsabar, Michael; Eapen, Vinay V; Mason, Jennifer M; Memisoglu, Gonen; Waterman, David P; Long, Marcus J; Bishop, Douglas K; Haber, James E

    2015-08-18

    In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5' to 3' end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5' to 3' resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

    Science.gov (United States)

    Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

  2. Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks.

    Science.gov (United States)

    Lee, Cindy; Banerjee, Tapahsama; Gillespie, Jessica; Ceravolo, Amanda; Parvinsmith, Matthew R; Starita, Lea M; Fields, Stanley; Toland, Amanda E; Parvin, Jeffrey D

    2015-12-01

    Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance. © 2015 WILEY PERIODICALS, INC.

  3. Rad50 zinc hook is important for the Mre11 complex to bind chromosomal DNA double-stranded breaks and initiate various DNA damage responses.

    Science.gov (United States)

    He, Jing; Shi, Linda Z; Truong, Lan N; Lu, Chi-Sheng; Razavian, Niema; Li, Yongjiang; Negrete, Alejandro; Shiloach, Joseph; Berns, Michael W; Wu, Xiaohua

    2012-09-14

    The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.

  4. Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses*

    Science.gov (United States)

    He, Jing; Shi, Linda Z.; Truong, Lan N.; Lu, Chi-Sheng; Razavian, Niema; Li, Yongjiang; Negrete, Alejandro; Shiloach, Joseph; Berns, Michael W.; Wu, Xiaohua

    2012-01-01

    The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn2+ promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation. PMID:22833675

  5. Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in the Candida albicans Diploid Genome.

    Science.gov (United States)

    Feri, Adeline; Loll-Krippleber, Raphaël; Commere, Pierre-Henri; Maufrais, Corinne; Sertour, Natacha; Schwartz, Katja; Sherlock, Gavin; Bougnoux, Marie-Elisabeth; d'Enfert, Christophe; Legrand, Mélanie

    2016-10-11

    The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive lethal allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in the C. albicans species. Recessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome

  6. Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double-strand breaks.

    Science.gov (United States)

    Tamura, Katsunori; Adachi, Yugo; Chiba, Keiko; Oguchi, Keiko; Takahashi, Hideo

    2002-03-01

    In higher organisms such as mammals and plants, DNA double-strand breaks (DSBs) are repaired preferentially by non-homologous end joining (NHEJ) rather than by homologous recombination. The NHEJ pathway is mediated by Ku, a heterodimer of approximately 70 and 80 kDa subunits, which contributes to various aspects of the metabolism of DNA ends in eukaryotic cells. On the basis of their predicted sequence similarity to human Ku70 and Ku80, cDNAs encoding the first plant homologues of these proteins (AtKu70 and AtKu80, respectively) have now been isolated from Arabidopsis thaliana. AtKu70 and AtKu80 share 28.6 and 22.5% amino acid sequence identity with human Ku70 and Ku80, respectively. Yeast two-hybrid analysis demonstrated that AtKu70 and AtKu80 form a heterodimer, and electrophoretic mobility-shift assays revealed that this heterodimer binds to double-stranded telomeric and non-telomeric DNA sequences, but not to single-stranded DNA. The AtKu heterodimer also possesses single-stranded DNA-dependent ATPase and ATP-dependent DNA helicase activities. Reverse transcription and the polymerase chain reaction revealed that AtKu70 and AtKu80 genes are expressed widely but at low levels in plant tissues. The expression of these two genes in cultured cells was markedly increased in response to the generation of DSBs by bleomycin or methylmethane sulfonate. These results suggest that the evolutionarily conserved Ku70-Ku80 heterodimer functions in DSB repair by the NHEJ pathway in A. thaliana.

  7. The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells.

    Science.gov (United States)

    Roth, Nadine; Klimesch, Jacqueline; Dukowic-Schulze, Stefanie; Pacher, Michael; Mannuss, Anja; Puchta, Holger

    2012-12-01

    In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  8. Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Woo; Xu, Guozhou; Persky, Nicole S.; Smogorzewska, Agata; Rudge, Derek G.; Buzovetsky, Olga; Elledge, Stephen J.; Pavletich, Nikola P. (Harvard-Med); (Cornell); (MSKCC)

    2011-08-29

    Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

  9. Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

    Energy Technology Data Exchange (ETDEWEB)

    W Joo; G Xu; n Persky; A Smogorzewska; D Rudge; O Buzovetsky; S Elledge; N Pavletich

    2011-12-31

    Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

  10. U. V. induces long-lived DNA breaks in Cockayne's syndrome and cells from an immunodeficient individual (46BR): defects and disturbance in post incision steps of excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Squires, S.; Johnson, R.T.

    1983-01-01

    In normal cells exposed to low U.V. doses the several enzymic steps of the excision repair process are closely coupled with the result that DNA gaps are transient and present at such low frequency that it is very difficult to detect them. Cells from a U.V.-sensitive human genetic disorder, Cockayne's Syndrome (CS) and from an immunodeficient individual 46BR, have been examined with respect to their incision capacity after U.V. in the presence and absence of inhibitors of DNA synthesis. We have measured the initial rates of DNA break accumulation in the presence of hydroxyurea and 1-beta-D arabinofuranosylcytosine and find that in both these groups the rate is only slightly lower than in normal cells. However, there is a marked difference between U.V. sensitive cells and normal in the accumulation of long-lived DNA breaks in the absence of inhibitors. While in normal cells practically no breaks could be detected, the U.V. sensitive cells accumulated significant numbers of DNA breaks within 15 min of incubation; 46BR cells showed almost the same level of DNA breaks without the inhibitors as with them. In CS break accumulation can be detected in the absence of inhibitors for only a short time after irradiation (approximately 30 min), but less so when deoxyribonucleosides are provided. The spontaneous break accumulation is related to the time elapsed since proteolytic detachment of the cells from monolayer; 24 h after replating CS breaks no longer accumulate in response to U.V. 46BR cells, on the other hand, accumulate breaks even 1 day after replating and express unligated gaps 2 h after irradiation with a relatively low U.V. dose such as 4 Jm-2. Provision of DNA precursors does not greatly reduce break accumulation. The extremely slow rate of gap sealing in 46BR cells is consistent with the hypothesis that a ligase defect is expressed in these cells.

  11. MRE11–RAD50–NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair

    Science.gov (United States)

    Roques, Céline; Coulombe, Yan; Delannoy, Mathieu; Vignard, Julien; Grossi, Simona; Brodeur, Isabelle; Rodrigue, Amélie; Gautier, Jean; Stasiak, Alicja Z; Stasiak, Andrzej; Constantinou, Angelos; Masson, Jean-Yves

    2009-01-01

    Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response. PMID:19609304

  12. Complex rectovaginal fistulas after pelvic organ prolapse repair with synthetic mesh: a multidisciplinary approach to evaluation and management.

    Science.gov (United States)

    Choi, Judy M; Nguyen, Vian; Khavari, Rose; Reeves, Keith; Snyder, Michael; Fletcher, Sophie G

    2012-01-01

    The use of synthetic mesh for transvaginal pelvic organ prolapse (POP) repair is associated with the rare complication of mesh erosion into hollow viscera. This study presents a single-institution series of complex rectovaginal fistulas (RVFs) after synthetic mesh-augmented POP repair, as well as strategies for identification and management. Institutional review board approval was obtained for this retrospective study. Data were collected and analyzed on all female patients undergoing RVF repair from 2000 to 2011 at our institution. Thirty-seven patients underwent RVF repair at our multidisciplinary center for restorative pelvic medicine. Of these, 10 (27.0%) were associated with POP repairs using mesh. The POP repairs resulting in RVF were transvaginal repair with mesh (n = 8), laparoscopic sacrocolpopexy with concomitant traditional posterior repair (n = 1), and robotic-assisted laparoscopic sacrocolpopexy (n = 1). Time to presentation was an average of 7.1 months after POP repair. Patients underwent a mean of 4.4 surgeries for definitive RVF repair, with 40% of patients requiring a bowel diversion (3 temporary ileostomies and 1 long-term colostomy). Mean follow-up time after last surgery was 9.2 months. On follow-up, 1 patient has a persistent fistula with vaginal mesh extrusion. One patient has persistent pelvic pain. This series highlights the significant impact of synthetic mesh complications in the posterior compartment. These complications should be cautionary for synthetic graft use by those with limited experience, particularly when an alternate choice of traditional repair is available. When symptoms of RVF are present, collaboration with a colon and rectal specialist should be initiated as soon as possible for evaluation and definitive repair.

  13. 3D Printing to Model Surgical Repair of Complex Congenitally Corrected Transposition of the Great Arteries.

    Science.gov (United States)

    Sahayaraj, R Anto; Ramanan, Sowmya; Subramanyan, Raghavan; Cherian, Kotturathu Mammen

    2017-01-01

    We report the use of three-dimensional (3D) modeling to plan surgery for physiologic repair of congenitally corrected transposition of the great arteries with pulmonary atresia, dextrocardia, and complex intra cardiac anatomy. Based on measurements made from the 3D printed model of the actual patient's anatomy, we anticipated using a composite valved conduit (Dacron tube graft, decellularized bovine jugular vein, and aortic homograft) to establish left ventricle-to-pulmonary artery continuity with relief of stenosis involving the pulmonary artery confluence and bilateral branch pulmonary arteries.

  14. Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in colon and liver of Big Blue rats: role of DNA adducts, strand breaks, DNA repair and oxidative stress

    DEFF Research Database (Denmark)

    Moller, P.; Wallin, H.; Vogel, U.

    2002-01-01

    weeks. There were dose-response relationships of DNA adducts (P-32-postlabeling) and DNA strand breaks (comet assay) in colon and liver tissues, with the highest levels of DNA adducts and strand breaks in the colon. There was dose-dependent induction of mutations in both the colon and the liver...... and colon. A lower frequency of mutations in the colon than in the liver could be related to higher expression of DNA repair enzymes in the former.......The contribution of oxidative stress, different types of DNA damage and expression of DNA repair enzymes in colon and liver mutagenesis induced by 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was investigated in four groups of six Big Blue rats fed diets with 0, 20, 70, and 200 mg IQ/kg for 3...

  15. Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci.

    Science.gov (United States)

    Jezkova, Lucie; Zadneprianetc, Mariia; Kulikova, Elena; Smirnova, Elena; Bulanova, Tatiana; Depes, Daniel; Falkova, Iva; Boreyko, Alla; Krasavin, Evgeny; Davidkova, Marie; Kozubek, Stanislav; Valentova, Olga; Falk, Martin

    2018-01-18

    Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored. We employed high-resolution confocal microscopy to examine phosphorylated histone H2AX (γH2AX)/p53-binding protein 1 (53BP1) focus streaks at the microscale level, focusing on the complexity, spatiotemporal behaviour and repair of DNA double-strand breaks generated by boron and neon ions accelerated at similar LET values (∼135 keV μm-1) and low energies (8 and 47 MeV per n, respectively). Cells were irradiated using sharp-angle geometry and were spatially (3D) fixed to maximize the resolution of these analyses. Both high-LET radiation types generated highly complex γH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET γ-rays. Surprisingly, neon ions produced even more complex γH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to γ-rays and boron ions eliminated a vast majority of foci (94% and 74%, respectively) within 24 h, 45% of the foci persisted in cells irradiated with neon. Our calculations suggest that the complexity of DSB damage critically depends on (increases with) the particle track core diameter. Thus, different particles with similar LET and energy may generate different types of DNA damage, which should be considered in future research.

  16. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

    Directory of Open Access Journals (Sweden)

    M Scott Brown

    2015-12-01

    Full Text Available The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs. Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt Rad51 filaments and also by one or more short Dmc1 filaments.

  17. Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore); Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore)

    2012-05-01

    Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are

  18. Staged hernia repair preceded by gastric bypass for the treatment of morbidly obese patients with complex ventral hernias.

    Science.gov (United States)

    Newcomb, W L; Polhill, J L; Chen, A Y; Kuwada, T S; Gersin, K S; Getz, S B; Kercher, K W; Heniford, B T

    2008-10-01

    Obesity may be the most predominant risk factor for recurrence following ventral hernia repair. This is secondary to significantly increased intra-abdominal pressures, higher rates of wound complications, and the technical difficulties encountered due to obesity. Medically managed weight loss prior to surgery is difficult. One potential strategy is to provide a surgical means to correct patient weight prior to hernia repair. After institutional review board approval, we reviewed the medical records of all patients who underwent gastric bypass surgery prior to the definitive repair of a complex ventral hernia at our medical center. Twenty-seven morbidly obese patients with an average of 3.7 (range 1-10) failed ventral hernia repairs underwent gastric bypass prior to definitive ventral hernia repair. Twenty-two of the gastric bypasses were open operations and five were laparoscopic. The patients' average pre-bypass body mass index (BMI) was 51 kg/m2 (range 39-69 kg/m2), which decreased to an average of 33 kg/m2 (range 25-37 kg/m2) at the time of hernia repair at a mean of 1.3 years (range 0.9-3.1 years) after gastric bypass. Seven patients had hernia repair at the same time as their gastric bypass (four sutured, three biologic mesh), all of which recurred. Of the 27 patients, 19 had an open hernia repair and eight had a laparoscopic repair. Panniculectomy was performed concurrently in 15 patients who had an open repair. Prior to formal hernia repair, one patient required an urgent operation to repair a hernia incarceration and a small-bowel obstruction 11 months after gastric bypass. The average hernia and mesh size was 203 cm2 (range 24-1,350 cm2) and 1,040 cm2 (range 400-2,700 cm2), respectively. There have been no recurrences at an average follow-up of 20 months (range 2 months-5 years). Gastric bypass prior to staged ventral hernia repair in morbidly obese patients with complex ventral hernias is a safe and definitive method to effect weight loss and facilitate a

  19. Mutant Cockayne syndrome group B protein inhibits repair of DNA topoisomerase I-DNA covalent complex.

    Science.gov (United States)

    Horibata, Katsuyoshi; Saijo, Masafumi; Bay, Mui N; Lan, Li; Kuraoka, Isao; Brooks, Philip J; Honma, Masamitsu; Nohmi, Takehiko; Yasui, Akira; Tanaka, Kiyoji

    2011-01-01

    Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B. © 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  20. Preoperative Botulinum toxin A enabling defect closure and laparoscopic repair of complex ventral hernia.

    Science.gov (United States)

    Rodriguez-Acevedo, Omar; Elstner, Kristen E; Jacombs, Anita S W; Read, John W; Martins, Rodrigo Tomazini; Arduini, Fernando; Wehrhahm, Michael; Craft, Colette; Cosman, Peter H; Dardano, Anthony N; Ibrahim, Nabeel

    2018-02-01

    Operative management of complex ventral hernia still remains a significant challenge for surgeons. Closure of large defects in the unprepared abdomen has serious pathophysiological consequences due to chronic contraction and retraction of the lateral abdominal wall muscles. We report outcomes of 56 consecutive patients who had preoperative Botulinum toxin A (BTA) abdominal wall relaxation facilitating closure and repair. This was a prospective observational study of 56 patients who underwent ultrasound-guided BTA into the lateral abdominal oblique muscles prior to elective ventral hernia repair between November 2012 and January 2017. Serial non-contrast abdominal CT imaging was performed to evaluate changes in lateral oblique muscle length and thickness. All hernias were repaired laparoscopically, or laparoscopic-open-laparoscopic (LOL) using intraperitoneal onlay mesh. 56 patients received BTA injections at predetermined sites to the lateral oblique muscles, which were well tolerated. Mean patient age was 59.7 years, and mean BMI was 30.9 kg/m 2 (range 21.8-54.0). Maximum defect size was 24 × 27 cm. A subset of 18 patients underwent preoperative pneumoperitoneum as an adjunct procedure. A comparison of pre-BTA to post-BTA imaging demonstrated an increase in mean lateral abdominal wall length from 16.1 cm to 20.1 cm per side, a mean gain of 4.0 cm/side (range 1.0-11.7 cm/side) (p LOL primary closure was achieved in all cases, with no clinical evidence of raised intra-abdominal pressures. One patient presented with a new fascial defect 26 months post-operative. Preoperative BTA to the lateral abdominal wall muscles is a safe and effective technique for the preparation of patients prior to operative management of complex ventral hernias. BTA temporary flaccid paralysis relaxes, elongates and thins the chronically contracted abdominal musculature. This in turn reduces lateral traction forces facilitating laparoscopic repair and fascial closure of large

  1. Preoperative progressive pneumoperitoneum complementing chemical component relaxation in complex ventral hernia repair.

    Science.gov (United States)

    Elstner, Kristen E; Read, John W; Rodriguez-Acevedo, Omar; Ho-Shon, Kevin; Magnussen, John; Ibrahim, Nabeel

    2017-04-01

    A rarely used technique for enabling closure of large ventral hernias with loss of domain is preoperative progressive pneumoperitoneum (PPP), which uses intermittent insufflation to gradually stretch the contracted abdominal wall muscles, increasing the capacity of the abdominal cavity. This allows the re-introduction of herniated viscera into the abdominal cavity and assists in closure of giant hernias which may otherwise be considered inoperable. This was a prospective study assessing 16 patients between 2013 and 2015 with multi-recurrent ventral hernia. All patients were treated preoperatively with both Botulinum Toxin A (BTA) injections to the lateral abdominal wall muscles to confer flaccid paralysis, and short-term PPP to passively expand the abdominal cavity. All patients underwent serial abdominal CT imaging, with pre- and post-treatment circumference measurements of the peritoneal cavity and hernia sac, prior to undergoing operative mesh repair of their hernia. The mean hernia defect size was 236 cm2, with mean 28 % loss of domain. The mean overall duration of PPP was 6.2 days. The mean gain in abdominal circumference was 4.9 cm (5.6 %) (p 0.002) after BTA and PPP. Fascial closure and mesh hernia repair were performed in all 16 patients, with no patients suffering from postoperative abdominal hypertension, ventilatory impairment, or wound dehiscence. There are no hernia recurrences to date. Eight patients (50 %) experienced PPP-related complications, consisting of subcutaneous emphysema, pneumothorax, pneumomediastinum, pneumocardium, and metabolic acidosis. No complication required intervention. PPP is a useful adjunct in the repair of complex ventral hernia. It passively expands the abdominal cavity, allowing viscera to re-establish right of domain. At the same time, it helps to minimize the risks of postoperative abdominal compartment syndrome and the sequelae of fascial closure under tension. However, its benefits must be carefully weighed with

  2. Breaking into the epithelial apical-junctional complex--news from pathogen hackers.

    Science.gov (United States)

    Vogelmann, Roger; Amieva, Manuel R; Falkow, Stanley; Nelson, W James

    2004-02-01

    The epithelial apical-junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical-junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical-junctional complex of the Ig superfamily--junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor--are important regulators of junction structure and function and represent critical targets of microbial virulence gene products.

  3. Breaking into the epithelial apical–junctional complex — news from pathogen hackers

    Science.gov (United States)

    Vogelmann, Roger; Amieva, Manuel R; Falkow, Stanley; Nelson, W James

    2012-01-01

    The epithelial apical–junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical–junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical–junctional complex of the Ig superfamily — junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor — are important regulators of junction structure and function and represent critical targets of microbial virulence gene products. PMID:15037310

  4. Lung transplantation and repair of complex congenital heart lesions in patients with pulmonary hypertension.

    Science.gov (United States)

    Mendeloff, E N; Huddleston, C B

    1998-04-01

    Pulmonary vascular disease in conjunction with either a previously repaired or an unrepaired congenital heart defect is the third most common indication for lung transplantation in the pediatric age range. Because scarcity of donor organs remains a critical issue and heart-lung donor blocks are becoming diminishingly available, efforts must be directed towards other options such as combining lung transplantation with correction of the underlying congenital heart defect. Certain defects like congenital pulmonary vein stenosis are eradicated by removal of the diseased lungs, whereas others such as complete atrioventricular canal and pulmonary atresia with ventricular septal defect require cardioplegic arrest of the heart and intracardiac repair in conjunction with the lung transplantation. A breakdown of this patient population into subgroups may be helpful both in thinking about the pathophysiology and in determining appropriate indications and timing of transplantation. Earlier studies from our center showed the high-risk nature and formidable undertaking of caring for this complex group of patients. Through continued experience, there has been gradual improvement in early outcomes. As with all other groups of lung transplantation patients, obliterative bronchiolitis remains the major deterrent to long-term survival.

  5. Catalysis vs. oxophilicity: Breaking the myth of inactive actinide-oxo complexes

    Energy Technology Data Exchange (ETDEWEB)

    Andrea, Tamer; Barnea, Eyal; Wang Jiaxi; Eisen, Moris S, E-mail: chmoris@tx.technion.ac.il [Schulich Faculty of Chemistry and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000 (Israel)

    2010-03-15

    For many decades, compounds containing oxygen atoms were excluded from the actinide-catalysis field because of the high oxophilic nature of these complexes. Pursuing the conceptual question about the potential activity of actinide-oxo bonds we were surprised to find that the coupling of aromatic aldehydes catalyzed by Cp*{sub 2}ThMe{sub 2} and Th(NEtMe){sub 4} via thorium-alkoxide intermediates take place in high yields to produce the corresponding esters. Here we present our breakthrough results including comprehensive mechanistic, deuterium labeling, kinetic and thermodynamic studies. In addition, the tetrachloride salt of uranium reacts with one equivalent of Li{sub 2}[(C{sub 5}Me{sub 4}){sub 2}SiMe{sub 2}] in DME to form the complex {l_brace}[{eta}{sup 5}-(C{sub 5}Me{sub 4}){sub 2}SiMe{sub 2}]UCl{sub 2{center_dot}}2LiCl{center_dot}2DME (13), which reacts with equimolar amounts of water in DME yielding the coordinative unsaturated bridged mono oxide and mono chloride uranium lithium salt complex {l_brace}[{eta}{sup 5}-(C{sub 5}Me{sub 4}){sub 2}SiMe{sub 2}]UCl{r_brace}{sub 2}(({mu}-O)(({mu}-Cl).Li(DME){sub 3}.DME (14). The alkylation of complexes 14 with BuLi gives the mono bridged dibutyl complex {l_brace}[{eta}{sup 5}-(C{sub 5}Me{sub 4}){sub 2}SiMe{sub 2}]UBu{r_brace}{sub 2}({mu}-O) (16). Complex 16 is an active catalyst for the disproportionation metathesis of TMSC{identical_to}CH and the cross-metathesis of TMSC{identical_to}CH or TMSC{identical_to}CTMS with various terminal alkynes. PhSiH{sub 3} reacts with complex 16 producing Ph{sub 2}SiH{sub 2} and SiH{sub 4} (caution) indicating the cleavage of the trimethylsilyl group from the phenyl moiety and the formation of uranium-silyl intermediate.

  6. Durham Smith Vest-Over-Pant Technique: Simple Procedure for a Complex Problem (Post-Hypospadias Repair Fistula).

    Science.gov (United States)

    Gite, Venkat A; Patil, Saurabh R; Bote, Sachin M; Siddiqui, Mohd Ayub Karam Nabi; Nikose, Jayant V; Kandi, Anitha J

    2017-01-01

    Urethrocutaneous fistula, which occurs after hypospadias surgery, is often a baffling problem and its treatment is challenging. The study aimed to evaluate the results of the simple procedure (Durham Smith vest-over-pant technique) for this complex problem (post-hypospadias repair fistula). During the period from 2011 to 2015, 20 patients with post-hypospadias repair fistulas underwent Durham Smith repair. Common age group was between 5 and 12 years. Site wise distribution of fistula was coronal 2 (10%), distal penile 7 (35%), mid-penile 7 (35%), and proximal-penile 4 (20%). Out of 20 patients, 15 had fistula of size 5 mm (25%). All cases were repaired with Durham Smith vest-over-pant technique by a single surgeon. In case of multiple fistulas adjacent to each other, all fistulas were joined to form single fistula and repaired. We have successfully repaired all post-hypospadias surgery urethrocutaneous fistulas using the technique described by Durham Smith with 100% success rate. Durham Smith vest-over-pant technique is a simple solution for a complex problem (post hypospadias surgery penile fistulas) in properly selected patients. © 2017 S. Karger AG, Basel.

  7. Breaking the stigma : the association between psychological factors and the complex regional pain syndrome

    OpenAIRE

    Beerthuizen, Annemerle

    2008-01-01

    textabstractIn 1900, Sudeck first described a post-traumatic pain syndrome with edema and trophic changes. This syndrome, known as Sudeck atrophy, was later called sympathetic reflex dystrophy and in 1994 renamed Complex Regional Pain Syndrome (CRPS). CRPS usually develops after a minor trauma such as an injury or fracture or after surgery, but spontaneous development of CRPS type 1 (CRPS1) has also been described. There are two types of CRPS described; CRPS1 arises without an obvious, detect...

  8. Suture welding for arthroscopic repair of peripheral triangular fibrocartilage complex tears.

    Science.gov (United States)

    Badia, Alejandro; Khanchandani, Prakash

    2007-03-01

    This report presents a method of arthroscopic repair of the peripheral triangular fibrocartilage tears by using ultrasonic suture welding technique, thus avoiding the need for traditional suture knots. This technique eliminates the potential causes of ulnar-sided wrist discomfort especially during the postoperative period. Twenty-three patients (9 women and 14 men; mean age, 35 years; range, 18-52 years) were operated during a 1-year period in 2001 for Palmer grade 1B triangular fibrocartilage complex tear and followed up for 17 months. At the final follow-up, the average wrist arc of motion was as follows: extension, 65 degrees; flexion, 56 degrees; supination, 80 degrees; pronation, 78 degrees; radial deviation, 12 degrees; and ulnar deviation, 25 degrees. Grip strength measured with a dynamometer (Jamar) averaged 81% of the contralateral side at the final evaluation (range, 53%-105%).

  9. Structure of UvrA nucleotide excision repair protein in complex with modified DNA

    Science.gov (United States)

    Jaciuk, Marcin; Nowak, Elżbieta; Skowronek, Krzysztof; Tańska, Anna; Nowotny, Marcin

    2012-01-01

    One of the primary pathways for removal of DNA damage is nucleotide excision repair (NER). In bacteria, the UvrA protein is the component of NER that locates the lesion. A notable feature of NER is its ability to act on many DNA modifications that vary in chemical structure. So far, the mechanism underlying this broad specificity has been unclear. Here, we report the first crystal structure of a UvrA protein in complex with a chemically modified oligonucleotide. The structure shows that the UvrA dimer does not contact the site of lesion directly, but rather binds the DNA regions on both sides of the modification. The DNA region harboring the modification is deformed, with the double helix bent and unwound. UvrA uses damage-induced deformations of the DNA and a less rigid structure of the modified double helix for indirect readout of the lesion. PMID:21240268

  10. DNA repair and global sumoylation are regulated by distinct Ubc9 noncovalent complexes.

    Science.gov (United States)

    Prudden, John; Perry, J Jefferson P; Nie, Minghua; Vashisht, Ajay A; Arvai, Andrew S; Hitomi, Chiharu; Guenther, Grant; Wohlschlegel, James A; Tainer, John A; Boddy, Michael N

    2011-06-01

    Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways.

  11. DNA Repair and Global Sumoylation Are Regulated by Distinct Ubc9 Noncovalent Complexes

    Science.gov (United States)

    Prudden, John; Perry, J. Jefferson P.; Nie, Minghua; Vashisht, Ajay A.; Arvai, Andrew S.; Hitomi, Chiharu; Guenther, Grant; Wohlschlegel, James A.; Tainer, John A.; Boddy, Michael N.

    2011-01-01

    Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways. PMID:21444718

  12. Breaking it down is better: haptic decomposition of complex movements aids in robot-assisted motor learning.

    Science.gov (United States)

    Klein, Julius; Spencer, Steven J; Reinkensmeyer, David J

    2012-05-01

    Training with haptic guidance has been proposed as a technique for learning complex movements in rehabilitation and sports, but it is unclear how to best deliver guidance-based training. Here, we hypothesized that breaking down a complex movement, similar to a tennis backhand, into simpler parts and then using haptic feedback from a robotic exoskeleton would help the motor system learn the movement. We also examined how the particular form of the decomposition affected learning. Three groups of unimpaired participants trained with the target arm movement broken down in three ways: 1) elbow flexion/extension and the unified shoulder motion independently ("anatomical" decomposition), 2) three component shoulder motions in Euler coordinates and elbow flexion/extension ("Euler" decomposition), or 3) the motion of the tip of the elbow and motion of the hand with respect to the elbow, independently ("visual" decomposition). A control group practiced the same number of movements, but experienced the target motion only, achieving eight times more direct practice with this motion. Despite less experience with the target motion, part training was better, but only when the arm trajectory was decomposed into anatomical components. Varying robotic movement training to include practice of simpler, anatomically-isolated motions may enhance its efficacy.

  13. Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

    DEFF Research Database (Denmark)

    Moss, Jennifer; Tinline-Purvis, Helen; Walker, Carol A

    2010-01-01

    Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found...

  14. Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

    NARCIS (Netherlands)

    K. Sugasawa (Kaoru); J.M.Y. Ng (Jessica); C. Masutani (Chikahide); T. Maekawa; A. Uchida; P.J. van der Spek (Peter); A.P.M. Eker (André); S. Rademakers (Suzanne); C.E. Visser (Cécile); A. Aboussekhra; R.D. Wood (Richard); F. Hanaoka (Fumio); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1997-01-01

    textabstractXPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a

  15. Evidence based on studies of the mus309 mutant, deficient in DNA double-strand break repair, that meiotic crossing over in Drosophila melanogaster is a two-phase process.

    Science.gov (United States)

    Portin, Petter

    2010-10-01

    The mus309 gene in Drosophila melanogaster encodes a RecQ helicase which is involved in DNA double-strand break (DSB) repair and specifically in the choice between the different pathways of the repair. In a brood pattern analysis of mus309 and wild type females which either had or had not experienced a temperature shock, different parameters of meiotic crossing over including map distances and crossover interference in the X chromosome were measured. The results suggest that, like in other eukaryotes studied, the control of meiotic crossover formation also in D. melanogaster is a two-phase process. The first phase seems to be temperature shock sensitive, independent of the mus309 gene and coincidental with the premeiotic DNA synthesis, thus most likely representing the formation of DSBs. The second phase seems to be temperature shock tolerant, dependent on the mus309 gene, occurring during the meiotic prophase and most likely representing the choice made by the oocyte between the different pathways of the DSB repair. A hypothesis of the localization of chiasmata is also presented, combining the mechanisms of interference and the so-called centromere effect, and based on the balance between the SDSA and DSBR pathways of DSB repair.

  16. Mineralization Induction of Gingival Fibroblasts and Construction of a Sandwich Tissue-Engineered Complex for Repairing Periodontal Defects.

    Science.gov (United States)

    Wu, Mingxuan; Wang, Jie; Zhang, Yanning; Liu, Huijuan; Dong, Fusheng

    2018-02-22

    BACKGROUND The ideal healing technique for periodontal tissue defects would involve the functional regeneration of the alveolar bone, cementum, and periodontal ligament, with new periodontal attachment formation. In this study, gingival fibroblasts were induced and a "sandwich" tissue-engineered complex (a tissue-engineered periodontal membrane between 2 tissue-engineered mineralized membranes) was constructed to repair periodontal defects. We evaluated the effects of gingival fibroblasts used as seed cells on the repair of periodontal defects and periodontal regeneration. MATERIAL AND METHODS Primitively cultured gingival fibroblasts were seeded bilaterally on Bio-Gide collagen membrane (a tissue-engineered periodontal membrane) or unilaterally on small intestinal submucosa segments, and their mineralization was induced. A tissue-engineered sandwich was constructed, comprising the tissue-engineered periodontal membrane flanked by 2 mineralized membranes. Periodontal defects in premolar regions of Beagles were repaired using the tissue-engineered sandwich or periodontal membranes. Periodontal reconstruction was compared to normal and trauma controls 10 or 20 days postoperatively. RESULTS Periodontal defects were completely repaired by the sandwich tissue-engineered complex, with intact new alveolar bone and cementum, and a new periodontal ligament, 10 days postoperatively. CONCLUSIONS The sandwich tissue-engineered complex can achieve ideal periodontal reconstruction rapidly.

  17. Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks.

    Science.gov (United States)

    Ling, Chen; Huang, Jing; Yan, Zhijiang; Li, Yongjiang; Ohzeki, Mioko; Ishiai, Masamichi; Xu, Dongyi; Takata, Minoru; Seidman, Michael; Wang, Weidong

    2016-01-01

    The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. Here, we successfully observed recruitment of FANCM at stalled forks using a newly developed protocol. We report that the FANCM recruitment depends upon its intrinsic DNA translocase activity, and its DNA-binding partner FAAP24. Moreover, it is dependent on the replication checkpoint kinase, ATR; but is independent of the FA core and FANCD2-FANCI complexes, two essential components of the FA pathway, indicating that the FANCM recruitment occurs downstream of ATR but upstream of the FA pathway. Interestingly, the recruitment of FANCM requires its direct interaction with Bloom syndrome complex composed of BLM helicase, Topoisomerase 3α, RMI1 and RMI2; as well as the helicase activity of BLM. We further show that the FANCM-BLM complex interaction is critical for replication stress-induced FANCM hyperphosphorylation, for normal activation of the FA pathway in response to ICLs, and for efficient traverse of ICLs by the replication machinery. Epistasis studies demonstrate that FANCM and BLM work in the same pathway to promote replication traverse of ICLs. We conclude that FANCM and BLM complex work together at stalled forks to promote both FA repair and replication traverse pathways of ICLs.

  18. Complexity Quantification for Overhead Transmission Line Emergency Repair Scheme via a Graph Entropy Method Improved with Petri Net and AHP Weighting Method

    Directory of Open Access Journals (Sweden)

    Jing Zhou

    2014-01-01

    Full Text Available According to the characteristics of emergency repair in overhead transmission line accidents, a complexity quantification method for emergency repair scheme is proposed based on the entropy method in software engineering, which is improved by using group AHP (analytical hierarchical process method and Petri net. Firstly, information structure chart model and process control flowchart model could be built by Petri net. Then impact factors on complexity of emergency repair scheme could be quantified into corresponding entropy values, respectively. Finally, by using group AHP method, weight coefficient of each entropy value would be given before calculating the overall entropy value for the whole emergency repair scheme. By comparing group AHP weighting method with average weighting method, experiment results for the former showed a stronger correlation between quantified entropy values of complexity and the actual consumed time in repair, which indicates that this new method is more valid.

  19. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    DEFF Research Database (Denmark)

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K

    2015-01-01

    DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised...... a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we...

  20. Femur fracture repair - discharge

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/patientinstructions/000166.htm Femur fracture repair - discharge To use the sharing features on this page, please enable JavaScript. You had a fracture (break) in the femur in your leg. It ...

  1. Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2000-02-01

    An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

  2. Breaking Bat

    Science.gov (United States)

    Aguilar, Isaac-Cesar; Kagan, David

    2013-01-01

    The sight of a broken bat in Major League Baseball can produce anything from a humorous dribbler in the infield to a frightening pointed projectile headed for the stands. Bats usually break at the weakest point, typically in the handle. Breaking happens because the wood gets bent beyond the breaking point due to the wave sent down the bat created…

  3. Availability,MTTF and Cost analysis of complex system under Preemptive resume repair policy using copula distribution

    Directory of Open Access Journals (Sweden)

    V V SINGH

    2014-10-01

    Full Text Available  In the present paper authors have focused  on the study of complex system consisting two subsystems, in series configuration and handling by a human operator. The subsystem (1 has three units at super priority, priority and ordinary & the subsystem (2 has one unit in series configuration with the subsystem-1. The whole system is operated by a human operator and human failure can also appear at different state where system is in operational mode. Initially super priority unit starts function and failure during  of super priority unit the priority unit start functioning and super priority unit goes under repair. The primitive resume repair policy is employed for repair of subsystem-1.The all failure rates are assumed to constants and follow exponential distribution but repair follow general and Gumbel-Hougaard family copula distribution. The system is studied by supplementary variable technique and Laplace transform. Various measure of reliability such as availability, state transition probabilities, mean time to system failure(M.T.T.F and profit function has been discussed for available maintenance  cost for all time and profit incurred by unit time for given interval. Some particular cases have been discussed for different values of different rates.

  4. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    Science.gov (United States)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  5. Efficacy of DNA double-strand breaks repair in breast cancer is decreased in carriers of the variant allele of the UBC9 gene c.73G>A polymorphism.

    Science.gov (United States)

    Synowiec, Ewelina; Krupa, Renata; Morawiec, Zbigniew; Wasylecka, Maja; Dziki, Lukasz; Morawiec, Jan; Blasiak, Janusz; Wozniak, Katarzyna

    2010-12-10

    UBC9 (E2) SUMO conjugating enzyme plays an important role in the maintenance of genome stability and integrity. In the present work we examined the association between the c.73G>A (Val25Met) polymorphism of the UBC9 gene (rs11553473) and efficacy of DNA double-strand breaks (DSBs) repair (DRE) in breast cancer patients. We determined the level of endogenous (basal) and exogenous (induced by γ-irradiation) DSBs and efficacy of their repair in peripheral blood lymphocytes of 57 breast cancer patients and 70 healthy individuals. DNA damage and repair were studied by neutral comet assay. Genotypes were determined in DNA from peripheral blood lymphocytes by allele-specific PCR (ASO-PCR). We also correlated genotypes with the clinical characteristics of breast cancer patients. We observed a strong association between breast cancer occurrence and the variant allele carried genotypes in patients with elevated level of basal as well as induced DNA damage (OR 6.74, 95% CI 2.27-20.0 and OR 5.33, 95% CI 1.81-15.7, respectively). We also found statistically significant (pA polymorphism of the UBC9 gene in breast cancer patients. Carriers of variant allele have decreased DNA DRE as compared to wild type genotype carriers. We did not find any association with the UBC9 gene polymorphism and estrogen and progesterone receptor status. The variant allele of the UBC9 gene polymorphism was strongly inversely related to HER negative breast cancer patients (OR 0.03, 95% CI 0.00-0.23). Our results suggest that the c.73G>A polymorphism of the UBC9 gene may affect DNA DSBs repair efficacy in breast cancer patients. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Efficacy of DNA double-strand breaks repair in breast cancer is decreased in carriers of the variant allele of the UBC9 gene c.73G>A polymorphism

    Energy Technology Data Exchange (ETDEWEB)

    Synowiec, Ewelina [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Krupa, Renata [Laboratory of DNA Repair, Department of Molecular Genetics, University of Lodz, Banacha 12/16, Lodz (Poland); Morawiec, Zbigniew; Wasylecka, Maja [Department of Surgical Oncology, N. Copernicus Hospital, Lodz (Poland); Dziki, Lukasz; Morawiec, Jan [Department of General and Colorectal Surgery, Medical University of Lodz, Lodz (Poland); Blasiak, Janusz [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Wozniak, Katarzyna, E-mail: wozniak@biol.uni.lodz.pl [Laboratory of DNA Repair, Department of Molecular Genetics, University of Lodz, Banacha 12/16, Lodz (Poland)

    2010-12-10

    UBC9 (E2) SUMO conjugating enzyme plays an important role in the maintenance of genome stability and integrity. In the present work we examined the association between the c.73G>A (Val25Met) polymorphism of the UBC9 gene (rs11553473) and efficacy of DNA double-strand breaks (DSBs) repair (DRE) in breast cancer patients. We determined the level of endogenous (basal) and exogenous (induced by {gamma}-irradiation) DSBs and efficacy of their repair in peripheral blood lymphocytes of 57 breast cancer patients and 70 healthy individuals. DNA damage and repair were studied by neutral comet assay. Genotypes were determined in DNA from peripheral blood lymphocytes by allele-specific PCR (ASO-PCR). We also correlated genotypes with the clinical characteristics of breast cancer patients. We observed a strong association between breast cancer occurrence and the variant allele carried genotypes in patients with elevated level of basal as well as induced DNA damage (OR 6.74, 95% CI 2.27-20.0 and OR 5.33, 95% CI 1.81-15.7, respectively). We also found statistically significant (p < 0.05) difference in DRE related to the c.73G>A polymorphism of the UBC9 gene in breast cancer patients. Carriers of variant allele have decreased DNA DRE as compared to wild type genotype carriers. We did not find any association with the UBC9 gene polymorphism and estrogen and progesterone receptor status. The variant allele of the UBC9 gene polymorphism was strongly inversely related to HER negative breast cancer patients (OR 0.03, 95% CI 0.00-0.23). Our results suggest that the c.73G>A polymorphism of the UBC9 gene may affect DNA DSBs repair efficacy in breast cancer patients.

  7. A UV-Induced Genetic Network Links the RSC Complex to Nucleotide Excision Repair and Shows Dose-Dependent Rewiring

    Directory of Open Access Journals (Sweden)

    Rohith Srivas

    2013-12-01

    Full Text Available Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions among 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

  8. Unsuitability of lymphoblastoid cell lines as surrogate of cryopreserved isolated lymphocytes for the analysis of DNA double-strand break repair activity

    Energy Technology Data Exchange (ETDEWEB)

    Zijno, Andrea [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Porcedda, Paola [Department of Clinical and Biological Sciences, University of Turin (Italy); Saini, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Allione, Alessandra [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Garofalo, Bruno; Marcon, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Guarrera, Simonetta [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Turinetto, Valentina; Minieri, Valentina [Department of Clinical and Biological Sciences, University of Turin (Italy); Funaro, Ada [Department of Genetics, Biology and Biochemistry, University of Turin (Italy); Crebelli, Riccardo [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Giachino, Claudia [Department of Clinical and Biological Sciences, University of Turin (Italy); Matullo, Giuseppe, E-mail: giuseppe.matullo@unito.it [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Department of Genetics, Biology and Biochemistry, University of Turin (Italy)

    2010-02-03

    As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by {gamma}-rays and DNA repair capacity were evaluated in unstimulated (G{sub 0}) and mitogen-simulated (G{sub 2}) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G{sub 0}-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G{sub 2}-treated PBMC compared to LCL. Concerning {gamma}-H2AX measurements, phosphorylation levels 1 h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of {gamma}-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype

  9. Structural zinc(II thiolate complexes relevant to the modeling of Ada repair protein: Application toward alkylation reactions

    Directory of Open Access Journals (Sweden)

    Mohamed M. Ibrahim

    2014-11-01

    Full Text Available The TtZn(II-bound perchlorate complex [TtZn–OClO3] 1 (Ttxyly = hydrotris[N-xylyl-thioimidazolyl]borate was used for the synthesis of zinc(II-bound ethanthiothiol complex [TtZn–SCH2CH3] 2 and its hydrogen-bond containing analog Tt–ZnSCH2CH2–NH(COOC(CH33 3. These thiolate complexes were examined as structural models for the active sites of Ada repair protein toward methylation reactions. The Zn[S3O] coordination sphere in complex 1 includes three thione donors from the ligand Ttixyl and one oxygen donor from the perchlorate coligand in ideally tetrahedral arrangement around the zinc center. The average Zn(1–S(thione bond length is 2.344 Å, and the Zn(1–O(1 bond length is 1.917 Å.

  10. Endovascular Retrieval of Entrapped Elephant Trunk Graft During Complex Hybrid Aortic Arch Repair

    Energy Technology Data Exchange (ETDEWEB)

    Damodharan, Karthikeyan, E-mail: drdkarthik@hotmail.com [Singapore General Hospital, Department of Diagnostic Radiology (Singapore); Chao, Victor T. T., E-mail: victor.chao.t.t@singhealth.com.sg [National Heart Centre, Department of Cardiothoracic Surgery (Singapore); Tay, Kiang Hiong, E-mail: tay.kiang.hiong@singhealth.com.sg [Singapore General Hospital, Department of Diagnostic Radiology (Singapore)

    2016-12-15

    Entrapment of the elephant trunk graft within the false lumen is a rare complication of surgical repair of an aortic dissection. This is normally retrieved by emergent open surgery. We describe a technique of endovascular retrieval of the dislodged graft, during hybrid aortic arch repair. The elephant trunk was cannulated through and through from a femoral access and the free end of the wire was snared and retrieved from a brachial access. The wire was externalised from both accesses and was used to reposition the graft into the true lumen using a body flossing technique.

  11. Biventricular Repair after Bilateral Pulmonary Artery Banding as a Rescue Procedure for a Neonate with Hypoplastic Left Heart Complex

    Directory of Open Access Journals (Sweden)

    Jae Kwang Yun

    2016-04-01

    Full Text Available Hypoplastic left heart complex (HLHC consists of less severe underdevelopment of the left ventricle without intrinsic left valvular stenosis, i.e., a subset of hypoplastic left heart syndrome (HLHS. HLHC patients may be able to undergo biventricular repair, while HLHS requires single ventricle palliation (or transplant. However, there is no consensus regarding the likelihood of favorable outcomes in neonates with HLHC selected to undergo this surgical approach. This case report describes a neonate with HLHC, co-arctation of the aorta (CoA, and patent ductus arteriosus (PDA who was initially palliated using bilateral pulmonary artery banding due to unstable ductus-dependent circulation. A postoperative echocardiogram showed newly appearing CoA and progressively narrowing PDA, which resulted in the need for biventricular repair 21 days following the palliation surgery. The patient was discharged on postoperative day 13 without complications and is doing clinically well seven months after surgery.

  12. Biventricular Repair after Bilateral Pulmonary Artery Banding as a Rescue Procedure for a Neonate with Hypoplastic Left Heart Complex

    Science.gov (United States)

    Yun, Jae Kwang; Bang, Ji Hyun; Kim, Young Hwee; Goo, Hyun Woo; Park, Jeong-Jun

    2016-01-01

    Hypoplastic left heart complex (HLHC) consists of less severe underdevelopment of the left ventricle without intrinsic left valvular stenosis, i.e., a subset of hypoplastic left heart syndrome (HLHS). HLHC patients may be able to undergo biventricular repair, while HLHS requires single ventricle palliation (or transplant). However, there is no consensus regarding the likelihood of favorable outcomes in neonates with HLHC selected to undergo this surgical approach. This case report describes a neonate with HLHC, co-arctation of the aorta (CoA), and patent ductus arteriosus (PDA) who was initially palliated using bilateral pulmonary artery banding due to unstable ductus-dependent circulation. A postoperative echocardiogram showed newly appearing CoA and progressively narrowing PDA, which resulted in the need for biventricular repair 21 days following the palliation surgery. The patient was discharged on postoperative day 13 without complications and is doing clinically well seven months after surgery. PMID:27064769

  13. Reconstruction of the symphysis pubis to repair a complex midline hernia in the setting of congenital bladder exstrophy.

    Science.gov (United States)

    Kohler, J E; Friedstat, J S; Jacobs, M A; Voelzke, B B; Foy, H M; Grady, R W; Gruss, J S; Evans, H L

    2015-08-01

    A 40-year-old man with congenital midline defect and wide pubic symphysis diastasis secondary to bladder exstrophy presented with a massive incisional hernia resulting from complications of multiple prior abdominal repairs. Using a multi-disciplinary team of general, plastic, and urologic surgeons, we performed a complex hernia repair including creation of a pubic symphysis with rib graft for inferior fixation of mesh. The skin graft overlying the peritoneum was excised, and the posterior rectus sheath mobilized, then re-approximated. The previously augmented bladder and urethra were mobilized into the pelvis, after which a rib graft was constructed from the 7th rib and used to create a symphysis pubis using a mortise joint. This rib graft was used to fix the inferior portion of a 20 × 25 cm porcine xenograft mesh in a retro-rectus position. With the defect closed, prior skin scars were excised and the wound closed over multiple drains. The patient tolerated the procedure well. His post-operative course was complicated by a vesico-cutaneous fistula and associated urinary tract and wound infections. This resolved by drainage with a urethral catheter and bilateral percutaneous nephrostomies. The patient has subsequently healed well with an intact hernia repair. The increased intra-abdominal pressure from his intact abdominal wall has been associated with increased stress urinary incontinence. Although a difficult operation prone to serious complications, reconstruction of the symphysis pubis is an effective means for creating an inferior border to affix mesh in complex hernia repairs associated with bladder exstrophy.

  14. Break-induced telomere synthesis underlies alternative telomere maintenance.

    Science.gov (United States)

    Dilley, Robert L; Verma, Priyanka; Cho, Nam Woo; Winters, Harrison D; Wondisford, Anne R; Greenberg, Roger A

    2016-11-03

    Homology-directed DNA repair is essential for genome maintenance through templated DNA synthesis. Alternative lengthening of telomeres (ALT) necessitates homology-directed DNA repair to maintain telomeres in about 10-15% of human cancers. How DNA damage induces assembly and execution of a DNA replication complex (break-induced replisome) at telomeres or elsewhere in the mammalian genome is poorly understood. Here we define break-induced telomere synthesis and demonstrate that it utilizes a specialized replisome, which underlies ALT telomere maintenance. DNA double-strand breaks enact nascent telomere synthesis by long-tract unidirectional replication. Proliferating cell nuclear antigen (PCNA) loading by replication factor C (RFC) acts as the initial sensor of telomere damage to establish predominance of DNA polymerase δ (Pol δ) through its POLD3 subunit. Break-induced telomere synthesis requires the RFC-PCNA-Pol δ axis, but is independent of other canonical replisome components, ATM and ATR, or the homologous recombination protein Rad51. Thus, the inception of telomere damage recognition by the break-induced replisome orchestrates homology-directed telomere maintenance.

  15. Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast.

    Directory of Open Access Journals (Sweden)

    Nina V Romanova

    2013-10-01

    Full Text Available DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair.

  16. Characterization of 26 deletion CNVs reveals the frequent occurrence of micro-mutations within the breakpoint-flanking regions and frequent repair of double-strand breaks by templated insertions derived from remote genomic regions.

    Science.gov (United States)

    Wang, Ye; Su, Peiqiang; Hu, Bin; Zhu, Wenjuan; Li, Qibin; Yuan, Ping; Li, Jiangchao; Guan, Xinyuan; Li, Fucheng; Jing, Xiangyi; Li, Ru; Zhang, Yongling; Férec, Claude; Cooper, David N; Wang, Jun; Huang, Dongsheng; Chen, Jian-Min; Wang, Yiming

    2015-06-01

    Copy number variations (CNVs) have increasingly been reported to cause, or predispose to, human disease. However, a large fraction of these CNVs have not been accurately characterized at the single-base-pair level, thereby hampering a better understanding of the mutational mechanisms underlying CNV formation. Here, employing a composite pipeline method derived from various inference-based programs, we have characterized 26 deletion CNVs [including three novel pathogenic CNVs involving an autosomal gene (EXT2) causing hereditary osteochondromas and an X-linked gene (CLCN5) causing Dent disease, as well as 23 CNVs previously identified by inference from a cohort of Canadian autism spectrum disorder families] to the single-base-pair level of accuracy from whole-genome sequencing data. We found that breakpoint-flanking micro-mutations (within 22 bp of the breakpoint) are present in a significant fraction (5/26; 19%) of the deletion CNVs. This analysis also provided evidence that a recently described error-prone form of DNA repair (i.e., repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome) not only causes human genetic disease but also impacts on human genome evolution. Our findings illustrate the importance of precise CNV breakpoint delineation for understanding the underlying mutational mechanisms and have implications for primer design in relation to the detection of deletion CNVs in clinical diagnosis.

  17. Xeroderma pigmentosum-Cockayne syndrome complex in two patients: absence of skin tumors despite severe deficiency of DNA excision repair.

    Science.gov (United States)

    Scott, R J; Itin, P; Kleijer, W J; Kolb, K; Arlett, C; Muller, H

    1993-11-01

    Two brothers had a complex combination of two DNA repair disorders: Cockayne syndrome and xeroderma pigmentosum. This rare combination has previously been observed in only two other patients. The clinical signs shared by these two brothers and the two other previously described patients include severe sun sensitivity, freckling, diminished stature, hearing and movement impairment, and neurologic degeneration. Although defective UV-induced unscheduled DNA synthesis has been demonstrated (5% of normal), no skin cancers have appeared in these 38- and 41-year-old brothers, whereas skin cancers developed at a relatively early age in the two previously described patients who also had defective UV-induced unscheduled DNA synthesis.

  18. Interactions involving the human RNA polymerase II transcription/nucleotide excision repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group B (CSB) protein.

    Science.gov (United States)

    Iyer, N; Reagan, M S; Wu, K J; Canagarajah, B; Friedberg, E C

    1996-02-20

    The human basal transcription factor TFIIH plays a central role in two distinct processes. TFIIH is an obligatory component of the RNA polymerase II (RNAP II) transcription initiation complex. Additionally, it is believed to be the core structure around which some if not all the components of the nucleotide excision repair (NER) machinery assemble to constitute a nucleotide excision repairosome. At least two of the subunits of TFIIH (XPB and XPD proteins) are implicated in the disease xeroderma pigmentosum (XP). We have exploited the availability of the cloned XPB, XPD, p62, p44, and p34 genes (all of which encode polypeptide subunits of TFIIH) to examine interactions between in vitro-translated polypeptides by co-immunoprecipitation. Additionally we have examined interactions between TFIIH components, the human NER protein XPG, and the CSB protein which is implicated in Cockayne syndrome (CS). Our analyses demonstrate that the XPB, XPD, p44, and p62 proteins interact with each other. XPG protein interacts with multiple subunits of TFIIH and with CSB protein.

  19. Complex ventral hernia repair using components separation with or without biologic mesh: a cost-utility analysis.

    Science.gov (United States)

    Chatterjee, Abhishek; Krishnan, Naveen M; Rosen, Joseph M

    2015-04-01

    A complex ventral hernia requiring abdominal wall reconstruction presents a challenging scenario to the surgeon. The use of biologic mesh in addition to performing a components separation (CS) is controversial. Our goal was to perform the first cost-utility analysis on the use of biologic mesh in addition to performing CS when performing complex ventral hernia repair. A comprehensive literature review was conducted to identify published complication and recurrence rates for ventral hernia repairs requiring CS with or without biologic mesh. The probabilities of the most common complications were combined with Medicare Current Procedural Terminology reimbursement codes, diagnosis related group reimbursement codes, and expert utility estimates to fit into a decision model to evaluate the cost utility of CS with and without biologic mesh in reconstructing ventral hernias. The decision model revealed a baseline cost increase of $775.65 and a 0.0517 increase in the quality-adjusted life-years when using biologic mesh yielding an incremental cost-utility ratio of $15,002.90/quality-adjusted life-year. One-way sensitivity analysis revealed that using biologic mesh was cost-effective using Medicare reimbursement rates but not at retail costs. The maximum price of biologic mesh to be cost-effective was $1813.53. The cost utility of biologic mesh when used with CS in ventral hernia repair is dependent on the financial perspective. It is cost-ineffective for hospitals and physicians paying retail costs but cost-effective for third-party payers providing Medicare reimbursement.

  20. Bypass of a 5′,8-cyclopurine-2′-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks

    Science.gov (United States)

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E.; Terzidis, Michael A.; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-01-01

    5′,8-cyclopurine-2′-deoxynucleosides including 5′,8-cyclo-dA (cdA) and 5′,8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5′,8-cyclopurine-2′-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5′R-cdA, but inefficiently bypassed a 5′S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5′,8-cyclopurine-2′-deoxynucleoside. Moreover, we found that bypass of a 5′S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5′,8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. PMID:26123757

  1. Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes.

    Science.gov (United States)

    Borthiry, Griselda R; Antholine, William E; Myers, Judith M; Myers, Charles R

    2008-08-01

    Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.

  2. DNA mismatch repair complex MutSβ promotes GAA·TTC repeat expansion in human cells.

    Science.gov (United States)

    Halabi, Anasheh; Ditch, Scott; Wang, Jeffrey; Grabczyk, Ed

    2012-08-24

    While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future.

  3. DNA Mismatch Repair Complex MutSβ Promotes GAA·TTC Repeat Expansion in Human Cells*

    Science.gov (United States)

    Halabi, Anasheh; Ditch, Scott; Wang, Jeffrey; Grabczyk, Ed

    2012-01-01

    While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future. PMID:22787155

  4. Genomic Approaches to DNA repair and Mutagenesis

    OpenAIRE

    Wyrick, John J.; Roberts, Steven A.

    2015-01-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processin...

  5. JMJD1C demethylates MDC1 to regulate the RNF8 and BRCA1-mediated chromatin response to DNA breaks

    DEFF Research Database (Denmark)

    Watanabe, Sugiko; Watanabe, Kenji; Akimov, Vyacheslav

    2013-01-01

    Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80-BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80-BRCA1 branch of this DNA...

  6. 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......-induced breaks are recognized by Rfa1 only after the cell enters S phase. This difference is dependent on the DNA end-binding Yku70/Yku80 complex. Cell-cycle regulation is also observed in the DNA damage checkpoint response. Specifically, the 9-1-1 complex is required in G1 cells to recruit the Ddc2 checkpoint...

  7. Outcomes and complications associated with perfluoro-n-octane and perfluoroperhydrophenanthrene in complex retinal detachment repair.

    Science.gov (United States)

    Scott, I U; Murray, T G; Flynn, H W; Smiddy, W E; Feuer, W J; Schiffman, J C

    2000-05-01

    To compare rates of perfluorocarbon liquid (PFCL) intraocular retention, anatomic and visual acuity outcomes, and complications associated with intraoperative perfluoro-n-octane (Perfluoron) versus perfluoroperhydrophenanthrene (Vitreon) in retinal detachment repair. Retrospective noncomparative consecutive case series. Records of consecutive patients who underwent retinal detachment repair with intraoperative Perfluoron (n = 78) or Vitreon (n = 84) at the Bascom Palmer Eye Institute between November 1, 1991, and October 31, 1994, were retrospectively reviewed. Comparison of PFCL intraocular retention rates, retinal reattachment rates, visual acuity outcomes, and postoperative complication rates between the Perfluoron and Vitreon groups at postoperative day 1, week 1, month 1, month 3, and month 6. To assess the study's generalizability, the Perfluoron data obtained in the current study were compared with results of the Perfluoron Multicenter Clinical Study. Retained Perfluoron was noted less frequently (P 25 mmHg) was 13 +/- 4% and 37 +/- 5%, respectively (P = 0.004). No significant difference was found between the groups in rates of postoperative hypotony. Results of this study are comparable with those observed in the Perfluoron Multicenter Clinical Study. In this study, Perfluoron was retained intraocularly less frequently than Vitreon. Although no significant difference was found between groups in retinal reattachment rates, Perfluoron is associated with slightly better 6-month visual acuity and lower rates of corneal abnormality and elevated intraocular pressure compared with Vitreon.

  8. Individual repair of radiation-induced DNA double-strand breaks in lymphocytes. Implications for radiation-induced dermatitis in breast cancer; Die individuelle Reparatur von strahleninduzierten DNA-Doppelstrangbruechen in Lymphozyten. Implikationen fuer die radiogene Dermatitis beim Mammakarzinom

    Energy Technology Data Exchange (ETDEWEB)

    Melchior, Patrick Wilhelm

    2011-07-01

    Purpose: Adjuvant 'whole breast radiotherapy' (WBRT) is the standard of care after breast conserving surgery in women with breast cancer. Throughout different cancer stages the addition of WBRT leads to significantly improved rates of freedom from local failure and overall survival. WBRT is generally well tolerated. A 5-10%-rate of severe acute or long-term side effects is commonly observed. For both radiation-mediated tumor-cell-elimination and induction of side effects, DNA-double-strand-breaks (DSB) presumably play the decisive role. The intensity of normal tissue reactions in radiotherapy can, in part, be attributed to the intrinsic DSB repair-capacity. In this study in vivo and in vitro experiments are carried through in order to assess DSB repair-kinetics in blood lymphocytes of women with breast cancer. These findings are to be correlated with the degree of radiation-induced normal tissue toxicity. Patients and Methods: Eighteen patients with breast cancer, in whom WBRT was indicated, were examined. A total WBRT dose of 50 Gy (single dose 2 Gy) with an additional boost-radiotherapy to the initial tumor-region to a total dose of 60-66 Gy was administered. DSB repair was determined by means of counting γ-H2AX foci in blood lymphocytes at predefined points in time, i.e. before and 0.5 h; 2.5 h; 5 h and 24 h after in vivo irradiation (1st fraction of WBRT) and before and 0.5 h; 2.5 h and 5 h after in vitro irradiation with increasing radiation doses in the range of 10 - 500 mGy. Acute normal tissue toxicity was scored on the basis of a modified RTOG-classification (main aspects were erythema and dry or moist skin desquamation). Results: DSB repair-halflife-times did not differ between patients with a higher or lower than average incidence of acute side effects. In patients with 'above average' side effects larger irradiation volumes were treated (volume surrounded by the 50%-isodose). Adjusted for these, no single patients showed elevated

  9. Radiation induced base excision repair (BER): a mechanistic mathematical approach.

    Science.gov (United States)

    Rahmanian, Shirin; Taleei, Reza; Nikjoo, Hooshang

    2014-10-01

    This paper presents a mechanistic model of base excision repair (BER) pathway for the repair of single-stand breaks (SSBs) and oxidized base lesions produced by ionizing radiation (IR). The model is based on law of mass action kinetics to translate the biochemical processes involved, step-by-step, in the BER pathway to translate into mathematical equations. The BER is divided into two subpathways, short-patch repair (SPR) and long-patch repair (LPR). SPR involves in replacement of single nucleotide via Pol β and ligation of the ends via XRCC1 and Ligase III, while LPR involves in replacement of multiple nucleotides via PCNA, Pol δ/ɛ and FEN 1, and ligation via Ligase I. A hallmark of IR is the production of closely spaced lesions within a turn of DNA helix (named complex lesions), which have been attributed to a slower repair process. The model presented considers fast and slow component of BER kinetics by assigning SPR for simple lesions and LPR for complex lesions. In the absence of in vivo reaction rate constants for the BER proteins, we have deduced a set of rate constants based on different published experimental measurements including accumulation kinetics obtained from UVA irradiation, overall SSB repair kinetic experiments, and overall BER kinetics from live-cell imaging experiments. The model was further used to calculate the repair kinetics of complex base lesions via the LPR subpathway and compared to foci kinetic experiments for cells irradiated with γ rays, Si, and Fe ions. The model calculation show good agreement with experimental measurements for both overall repair and repair of complex lesions. Furthermore, using the model we explored different mechanisms responsible for inhibition of repair when higher LET and HZE particles are used and concluded that increasing the damage complexity can inhibit initiation of LPR after the AP site removal step in BER. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Anti-inflammatory and osteogenesis of complex particles composed of ceric oxide and bioglass on repair of rabbit femoral defects

    Directory of Open Access Journals (Sweden)

    Hai HUANG

    2011-02-01

    Full Text Available Objective To observe the anti-inflammatory and osteogenesis of the complex particles composed of ceric oxide and bioglass implanted into rabbit femoral defects.Methods The bilateral penetrating femoral condylar defects of 6mm in diameter were made in 6 adult New Zealand White rabbits,bioglass particles and complex particles composed of ceric oxide and bioglass were randomly implanted into the left or right condylar defect.Animals were sacrificed at 2 and 4 weeks after operation(3 each,specimens from defect sites were harvested for histological examination to observe the inflammatory reaction and calculate the ratio of new bone and grafts on the defect area.Results No inflammatory reaction was found in the defect area filled with bioglass particles or complex particles composed of ceric oxide and bioglass.New bone was observed in the defects 2 weeks after operation,and no significant difference on the bone formation rate existed between the two materials(P > 0.05.The osteogenesis of both materials was more obvious in the 4th week than in the 2nd week(P 0.05.No obvious degradation of the grafts implanted into the defect area occurred in the 4th week compared with that in the 2nd week(P > 0.05.Conclusion The complex particles composed of ceric oxide and bioglass has anti-inflammatory activity and similar osteogenesis to normal bioglass particles,and may be used in the repair of bone defects.

  11. The complex dialogue between (myo)fibroblasts and the extracellular matrix during skin repair processes and ageing.

    Science.gov (United States)

    Vedrenne, N; Coulomb, B; Danigo, A; Bonté, F; Desmoulière, A

    2012-02-01

    The fibroblasts and the myofibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. The (myo)fibroblasts are embedded in a sophisticated extracellular matrix (ECM) that they secrete, and a complex and interactive dialogue exists between (myo)fibroblasts and their microenvironment. The composition of the ECM around (myo)fibroblasts is variable depending on the situation and, in addition to the secretion of the ECM, the (myo)fibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases can remodel this ECM. The (myo)fibroblasts and their microenvironment form a changing network with reciprocal actions leading to cell differentiation, proliferation, quiescence or apoptosis, and also acting on growth factor biodisponibility. In pathological situations (such as chronic wounds or excessive scarring), or during ageing, especially due to ultraviolet exposition, this dialogue between the (myo)fibroblasts and their microenvironment is disrupted, leading to repair defects or to skin injuries with unaesthetic alterations such as wrinkles. Knowing the intimate exchanges between the (myo)fibroblasts and their microenvironment represents a fascinating domain important not only for characterizing new targets and drugs able to prevent pathological developments but also for interfering with skin alterations observed during ageing. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  12. Primitive neuroectodermal tumor of the zygomaticoorbital complex: a rare location and ways of surgical repair of the area

    Directory of Open Access Journals (Sweden)

    Ch. R. Ragimov

    2015-01-01

    Full Text Available Primitive neuroectodermal tumor in the zygomaticoorbital region is a rare neoplasm of the head and neck. Due to the necessity for wide radical excision of a primary tumor, there may be serious functional and cosmetic disorders that substantially affect quality of life in patients. Restoration of this region is one of the challenges of reconstructive surgery because of the specific features of the relief of bone structures. The paper describes a clinical case of the site of primitive neuroectodermal tumor in the zygomaticoorbital complex and a method for repairing postresectional defect and completely recovering the function of the organ of vision and aesthetic parameters of the face.

  13. The Break

    DEFF Research Database (Denmark)

    Strand, Anete Mikkala Camille

    2018-01-01

    The chapter elaborates on how to deal with one of the major challenges facing organizations worldwide; Stress. The Break enacts a quantum approach to meet the challenges by proposing a combination of three different quantum storytelling technologies; protreptic mentoring, walking and material...... storytelling to enact fruitful breakings of patterns unbecoming. The claim being, that the hamster wheel of Work-life anno 2016 needs reconfiguration and the simple yet fruitful manner by which this is done is through acknowledging the benefits of bodies, spaces and artifacts – and the benefits of actually...... taking a break, discontinuing for a moment in order to continue better, wiser and more at ease. Both within and as part of the daily routines, and – now and then – outside these routines in the majesty of nature with time to explore and redirect the course of life in companionships with fellow man...

  14. DNA Repair and Global Sumoylation Are Regulated by Distinct Ubc9 Noncovalent Complexes

    OpenAIRE

    Prudden, John; Perry, J. Jefferson P.; Nie, Minghua; Ajay A Vashisht; Arvai, Andrew S.; Hitomi, Chiharu; Guenther, Grant; Wohlschlegel, James A; Tainer, John A.; Boddy, Michael N

    2011-01-01

    Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 p...

  15. Effect of surface conditioning modalities on the repair bond strength of resin composite to the zirconia core / veneering ceramic complex.

    Science.gov (United States)

    Ozcan, Mutlu; Valandro, Luiz Felipe; Pereira, Sarina Maciel; Amaral, Regina; Bottino, Marco Antonio; Pekkan, Gurel

    2013-06-01

    This study evaluated the effect of different surface conditioning protocols on the repair strength of resin composite to the zirconia core / veneering ceramic complex, simulating the clinical chipping phenomenon. Forty disk-shaped zirconia core (Lava Zirconia, 3M ESPE) (diameter: 3 mm) specimens were veneered circumferentially with a feldspathic veneering ceramic (VM7, Vita Zahnfabrik) (thickness: 2 mm) using a split metal mold. They were then embedded in autopolymerizing acrylic with the bonding surfaces exposed. Specimens were randomly assigned to one of the following surface conditioning protocols (n = 10 per group): group 1, veneer: 4% hydrofluoric acid (HF) (Porcelain Etch) + core: aluminum trioxide (50-µm Al2O3) + core + veneer: silane (ESPE-Sil); group 2: core: Al2O3 (50 µm) + veneer: HF + core + veneer: silane; group 3: veneer: HF + core: 30 µm aluminum trioxide particles coated with silica (30 µm SiO2) + core + veneer: silane; group 4: core: 30 µm SiO2 + veneer: HF + core + veneer: silane. Core and veneer ceramic were conditioned individually but no attempt was made to avoid cross contamination of conditioning, simulating the clinical intraoral repair situation. Adhesive resin (VisioBond) was applied to both the core and the veneer ceramic, and resin composite (Quadrant Posterior) was bonded onto both substrates using polyethylene molds and photopolymerized. After thermocycling (6000 cycles, 5°C-55°C), the specimens were subjected to shear bond testing using a universal testing machine (1 mm/min). Failure modes were identified using an optical microscope, and scanning electron microscope images were obtained. Bond strength data (MPa) were analyzed statistically using the non-parametric Kruskal-Wallis test followed by the Wilcoxon rank-sum test and the Bonferroni Holm correction (α = 0.05). Group 3 demonstrated significantly higher values (MPa) (8.6 ± 2.7) than those of the other groups (3.2 ± 3.1, 3.2 ± 3, and 3.1 ± 3.5 for groups 1, 2, and 4

  16. A Complex Network Theory Approach for the Spatial Distribution of Fire Breaks in Heterogeneous Forest Landscapes for the Control of Wildland Fires.

    Science.gov (United States)

    Russo, Lucia; Russo, Paola; Siettos, Constantinos I

    2016-01-01

    Based on complex network theory, we propose a computational methodology which addresses the spatial distribution of fuel breaks for the inhibition of the spread of wildland fires on heterogeneous landscapes. This is a two-level approach where the dynamics of fire spread are modeled as a random Markov field process on a directed network whose edge weights are determined by a Cellular Automata model that integrates detailed GIS, landscape and meteorological data. Within this framework, the spatial distribution of fuel breaks is reduced to the problem of finding network nodes (small land patches) which favour fire propagation. Here, this is accomplished by exploiting network centrality statistics. We illustrate the proposed approach through (a) an artificial forest of randomly distributed density of vegetation, and (b) a real-world case concerning the island of Rhodes in Greece whose major part of its forest was burned in 2008. Simulation results show that the proposed methodology outperforms the benchmark/conventional policy of fuel reduction as this can be realized by selective harvesting and/or prescribed burning based on the density and flammability of vegetation. Interestingly, our approach reveals that patches with sparse density of vegetation may act as hubs for the spread of the fire.

  17. A Complex Network Theory Approach for the Spatial Distribution of Fire Breaks in Heterogeneous Forest Landscapes for the Control of Wildland Fires.

    Directory of Open Access Journals (Sweden)

    Lucia Russo

    Full Text Available Based on complex network theory, we propose a computational methodology which addresses the spatial distribution of fuel breaks for the inhibition of the spread of wildland fires on heterogeneous landscapes. This is a two-level approach where the dynamics of fire spread are modeled as a random Markov field process on a directed network whose edge weights are determined by a Cellular Automata model that integrates detailed GIS, landscape and meteorological data. Within this framework, the spatial distribution of fuel breaks is reduced to the problem of finding network nodes (small land patches which favour fire propagation. Here, this is accomplished by exploiting network centrality statistics. We illustrate the proposed approach through (a an artificial forest of randomly distributed density of vegetation, and (b a real-world case concerning the island of Rhodes in Greece whose major part of its forest was burned in 2008. Simulation results show that the proposed methodology outperforms the benchmark/conventional policy of fuel reduction as this can be realized by selective harvesting and/or prescribed burning based on the density and flammability of vegetation. Interestingly, our approach reveals that patches with sparse density of vegetation may act as hubs for the spread of the fire.

  18. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting.

    Directory of Open Access Journals (Sweden)

    Woo-Hyun Chung

    2010-05-01

    Full Text Available The formation of single-stranded DNA (ssDNA at double-strand break (DSB ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2-4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR, reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Delta exo1Delta mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Delta exo1Delta, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Delta exo1Delta cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.

  19. Mechanical Stress Changes the Complex Interplay Between HO-1, Inflammation and Fibrosis, During Excisional Wound Repair.

    Science.gov (United States)

    Cremers, Niels A J; Suttorp, Maarten; Gerritsen, Marlous M; Wong, Ronald J; van Run-van Breda, Coby; van Dam, Gooitzen M; Brouwer, Katrien M; Kuijpers-Jagtman, Anne Marie; Carels, Carine E L; Lundvig, Ditte M S; Wagener, Frank A D T G

    2015-01-01

    Mechanical stress following surgery or injury can promote pathological wound healing and fibrosis, and lead to functional loss and esthetic problems. Splinted excisional wounds can be used as a model for inducing mechanical stress. The cytoprotective enzyme heme oxygenase-1 (HO-1) is thought to orchestrate the defense against inflammatory and oxidative insults that drive fibrosis. Here, we investigated the activation of the HO-1 system in a splinted and non-splinted full-thickness excisional wound model using HO-1-luc transgenic mice. Effects of splinting on wound closure, HO-1 promoter activity, and markers of inflammation and fibrosis were assessed. After seven days, splinted wounds were more than three times larger than non-splinted wounds, demonstrating a delay in wound closure. HO-1 promoter activity rapidly decreased following removal of the (epi)dermis, but was induced in both splinted and non-splinted wounds during skin repair. Splinting induced more HO-1 gene expression in 7-day wounds; however, HO-1 protein expression remained lower in the epidermis, likely due to lower numbers of keratinocytes in the re-epithelialization tissue. Higher numbers of F4/80-positive macrophages, αSMA-positive myofibroblasts, and increased levels of the inflammatory genes IL-1β, TNF-α, and COX-2 were present in 7-day splinted wounds. Surprisingly, mRNA expression of newly formed collagen (type III) was lower in 7-day wounds after splinting, whereas, VEGF and MMP-9 were increased. In summary, these data demonstrate that splinting delays cutaneous wound closure and HO-1 protein induction. The pro-inflammatory environment following splinting may facilitate higher myofibroblast numbers and increase the risk of fibrosis and scar formation. Therefore, inducing HO-1 activity against mechanical stress-induced inflammation and fibrosis may be an interesting strategy to prevent negative effects of surgery on growth and function in patients with orofacial clefts or in patients with

  20. Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.

    Science.gov (United States)

    Jiao, Chensong; Summerlin, Matthew; Bruzik, Karol S; Hanakahi, Leslyn

    2015-10-20

    Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems.

  1. The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex.

    Science.gov (United States)

    van Gool, A J; Citterio, E; Rademakers, S; van Os, R; Vermeulen, W; Constantinou, A; Egly, J M; Bootsma, D; Hoeijmakers, J H

    1997-10-01

    Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

  2. Salvage irrigation-suction in gracilis muscle repair of complex rectovaginal and rectourethral fistulas.

    Science.gov (United States)

    Chen, Xiao-Bing; Wang, You-Xin; Jiang, Hua; Liao, Dai-Xiang; Yu, Jun-Hui; Luo, Cheng-Hua

    2013-10-21

    To evaluate the efficacy of gracilis muscle transposition and postoperative salvage irrigation-suction in the treatment of complex rectovaginal fistulas (RVFs) and rectourethral fistulas (RUFs). Between May 2009 and March 2012, 11 female patients with complex RVFs and 8 male patients with RUFs were prospectively enrolled. Gracilis muscle transposition was undertaken in all patients and postoperative wound irrigation-suction was performed in patients with early leakage. Efficacy was assessed in terms of the success rate and surgical complications. SF-36 quality of life (QOL) scores and Wexner fecal incontinence scores were compared before and after surgery. The fistulas healed in 14 patients after gracilis muscle transposition; the initial healing rate was 73.7%. Postoperative leakage occurred and continuous irrigation-suction of wounds was undertaken in 5 patients: 4 healed and 1 failed, and postoperative fecal diversions were performed for the patient whose treatment failed. At a median follow-up of 17 mo, the overall healing rate was 94.7%. Postoperative complications occurred in 4 cases. Significant improvement was observed in the quality outcomes framework scores (P irrigation-suction-assisted healing group. Gracilis muscle transposition and postoperative salvage wound irrigation-suction gained a high success rate in the treatment of complex RVFs and RUFs. QOL and fecal incontinence were significantly improved after the successful healing of RVFs and RUFs.

  3. Structure of p15PAF-PCNA complex and implications for clamp sliding during DNA replication and repair

    DEFF Research Database (Denmark)

    De Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B

    2015-01-01

    The intrinsically disordered protein p15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP......-box) of p15(PAF) tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15(PAF) also contacts the inside of, and passes through, the PCNA ring. The disordered p15(PAF) termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both...... free and PCNA-bound p15(PAF) binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15(PAF) acts as a flexible drag that regulates PCNA sliding along...

  4. Structure of p15PAF-PCNA complex and implications for clamp sliding during DNA replication and repair

    Science.gov (United States)

    de Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B.; Molina, Rafael; Cordeiro, Tiago N.; Castillo, Francisco; Villate, Maider; Merino, Nekane; Delgado, Sandra; Gil-Cartón, David; Luque, Irene; Diercks, Tammo; Bernadó, Pau; Montoya, Guillermo; Blanco, Francisco J.

    2015-03-01

    The intrinsically disordered protein p15PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15PAF-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

  5. Structure of p15(PAF)-PCNA complex and implications for clamp sliding during DNA replication and repair.

    Science.gov (United States)

    De Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B; Molina, Rafael; Cordeiro, Tiago N; Castillo, Francisco; Villate, Maider; Merino, Nekane; Delgado, Sandra; Gil-Cartón, David; Luque, Irene; Diercks, Tammo; Bernadó, Pau; Montoya, Guillermo; Blanco, Francisco J

    2015-03-12

    The intrinsically disordered protein p15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15(PAF) tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15(PAF) also contacts the inside of, and passes through, the PCNA ring. The disordered p15(PAF) termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15(PAF) binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15(PAF) acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

  6. Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

    DEFF Research Database (Denmark)

    Akbari, Mansour; Otterlei, Marit; Pena Diaz, Javier

    2004-01-01

    , PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG......Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using...

  7. Localization of checkpoint and repair proteins in eukaryotes

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2005-01-01

    In eukaryotes, the cellular response to DNA damage depends on the type of DNA structure being recognized by the checkpoint and repair machinery. DNA ends and single-stranded DNA are hallmarks of double-strand breaks and replication stress. These two structures are recognized by distinct sets...... is largely controlled by a network of protein-protein interactions, with the Mre11 complex initiating assembly at DNA ends and replication protein A directing recruitment to single-stranded DNA. This review summarizes current knowledge on the cellular organization of DSB repair and checkpoint proteins...... focusing on budding yeast and mammalian cells....

  8. Electroweak breaking and supersymmetry breaking

    Indian Academy of Sciences (India)

    We discuss the clash between the absence of fine tuning in the Higgs potential and a sufficient suppression of flavour changing neutral current transitions in supersymmetric extensions of the standard model. It is pointed out that horizontal U ( 1 ) symmetry combined with the D -term supersymmetry breaking provides a ...

  9. Evidence that the Rad1 and Rad10 proteins of Saccharomyces cerevisiae participate as a complex in nucleotide excision repair of UV radiation damage.

    OpenAIRE

    Siede, W.; Friedberg, A S; Friedberg, E C

    1993-01-01

    A newly characterized rad1 missense mutation (rad1-20) in the yeast Saccharomyces cerevisiae maps to a region of the Rad1 polypeptide known to be required for Rad1-Rad10 complex formation. The UV sensitivity of the rad1-20 mutant can be partially and specifically corrected by overexpression of wild-type Rad10 protein. These results suggest that complex formation between the Rad1 and Rad10 proteins is required for nucleotide excision repair.

  10. The Break

    DEFF Research Database (Denmark)

    Strand, Anete Mikkala Camille; Larsen, Jens

    2015-01-01

    that language and the social has been granted too much power on the dispense of the bodily, physical and biological – or in short, in dispense of the material. The break To be or not to be poses the theoretical notion of dis-/continuity (Barad, 2007, 2010) from the quantum approach to storytelling (Strand 2012...... and euro each year. The paper tries to explore new ways to deal with these challenges through a quantum approach to storytelling where the enactment of core values, bodies, spaces and artifacts positions managers and CEO’s from major Scandinavian organizations in sites where they can re-evaluate their life...... stones on a table in an office of a municipality in Denmark. Silence….. Rebuilding rooms for taking breaks with the inclusion of different activities such as a game of soccer or a hike seems to provide the tools to rework these imbalances or enslaving patterns. Break……. The attempt at meeting...

  11. Patients in Whom Arthroscopic Bankart Repair is Not Enough: Evaluation and Management of Complex Anterior Glenohumeral Instability.

    Science.gov (United States)

    Tokish, John M; Lafosse, Laurent; Giacomo, Giovanni Di; Arciero, Robert

    2017-02-15

    Arthroscopic Bankart repair has become the most common treatment option for patients who have anterior shoulder instability. Although arthroscopic Bankart repair is generally an effective treatment method, it may be insufficient for the treatment of many patients who have anterior shoulder instability. Risk factors for failure of arthroscopic Bankart repair include younger age, level and type of sport, and shoulder specific risks, such as ligamentous laxity and the presence of bone loss. Recently, researchers have defined the limits of arthroscopic Bankart repair and more clearly defined treatment options for patients who have a high risk for failure after arthroscopic Bankart repair. Surgeons must recognize patients with anterior shoulder instability in whom a more aggressive surgical approach should be considered as well as patients who have a high risk for failure after arthroscopic Bankart repair to optimize outcomes in this patient population.

  12. Ability of sodium copper chlorophyllin complex to repair photoaged skin by stimulation of biomarkers in human extracellular matrix

    Directory of Open Access Journals (Sweden)

    McCook JP

    2016-07-01

    Full Text Available John P McCook,1 Thomas J Stephens,2 Lily I Jiang,2 Robert M Law,3 Vincent Gotz4 1Discovery Partners LLC, Frisco, 2Thomas J. Stephens & Associates, Inc., Richardson, 3ProPath, Dallas, TX, 4MDRejuvena, Inc., San Diego, CA, USA Purpose: To examine the effect of sodium copper chlorophyllin complex on the expression of biomarkers of photoaged dermal extracellular matrix indicative of skin repair.Patients and methods: Following a previously published 12-day clinical assessment model, skin biopsy samples from the forearms of four healthy females with signs of photoaged skin were obtained and samples were analyzed by immunohistochemistry for key biomarkers of aging skin after each subject was treated with a test material consisting of a gel containing a liposomal dispersion of sodium copper chlorophyllin complex 0.05%, a positive control of tretinoin cream 0.025%, and an untreated negative control.Results: There was a statistically significantly greater amount of fibrillin/amyloid P and epidermal mucins found for skin treated with the test material containing 0.05% sodium copper chlorophyllin complex and the reference control tretinoin 0.025% cream compared to the negative control (untreated site. Expression of procollagen 1 and dermal mucin also showed a greater presence in the samples treated with the test material and the reference control compared to the negative control, though the differences were not statistically significant. No adverse events were observed or reported by the subjects during the course of the study.Conclusion: The results of this human biopsy study suggest that both retinoids and sodium copper chlorophyllin complex have beneficial effects on biomarkers of photoaged skin. Products containing both sodium copper chlorophyllin complex and retinols may provide a dual approach to reversing age-related decreases in hyaluronic acid (HA in the skin: inhibition of the breakdown of HA via sodium copper chlorophyllin complex by inhibition

  13. Risk-adjusted adverse outcomes in complex abdominal wall hernia repair with biologic mesh: A case series of 140 patients.

    Science.gov (United States)

    Latifi, Rifat; Samson, David; Haider, Ansab; Azim, Asad; Iftikhar, Hajira; Joseph, Bellal; Tilley, Elizabeth; Con, Jorge; Gashi, Saranda; El-Menyar, Ayman

    2017-07-01

    Biologic mesh is preferred for repair of complex abdominal wall hernias (CAWHs) in patients at high risk of wound infection. We aimed to identify predictors of adverse outcomes after complex abdominal wall hernia repair (CAWR) using biologic mesh with different placement techniques and under different surgical settings. A retrospective case series study was conducted on all patients who underwent CAWR with biologic mesh between 2010 and 2015 at a tertiary medical center. the study population included 140 patients with a mean age of 54 ± 14 years and a median follow up period 8.8 months. Mesh size ranged from 50 to 1225 cm2. Ninety percent of patients had undergone previous surgery. Type of surgery was classified as elective in 50.7%, urgent in 24.3% and emergent in 25.0% and a porcine mesh was implanted in 82.9%. The most common mesh placement technique was underlay (70.7%), followed by onlay (16.4%) and bridge (12.9%). Complications included wound complications (30.7%), reoperation (25.9%), hernia recurrence (20.7%), and mesh removal (10.0%). Thirty-two patients (23.0%) were admitted to the ICU and the mean hospital length of stay was 10.8 ± 17.5 days. Age-sex adjusted predictors of recurrence were COPD (OR 4.2; 95%CI 1.003-17.867) and urgent surgery (OR 10.5; 95%CI 1.856-59.469), whereas for reoperation, mesh size (OR 6.8; 95%CI 1.344-34.495) and urgent surgery (OR 5.2; 95%CI 1.353-19.723) were the predictors. Using biologic mesh, one-quarter and one-fifth of CAWR patients are complicated with reoperation or recurrence, respectively. The operation settings and comorbidity may play a role in these outcomes regardless of the mesh placement techniques. Copyright © 2017 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.

  14. Effects of genetic changes to the begomovirus/betasatellite complex causing cotton leaf curl disease in South Asia post-resistance breaking.

    Science.gov (United States)

    Briddon, Rob W; Akbar, Fazal; Iqbal, Zafar; Amrao, Luqman; Amin, Imran; Saeed, Muhammad; Mansoor, Shahid

    2014-06-24

    Cotton leaf curl disease (CLCuD) has been a problem for cotton production across Pakistan and north-eastern India since the early 1990s. The appearance of the disease has been attributed to the introduction, and near monoculture of highly susceptible cotton varieties. During the intervening period the genetic make-up of the virus(es) causing the disease has changed dramatically. The most prominent of these changes has been in response to the introduction of CLCuD-resistant cotton varieties in the late 1990s, which provided a brief respite from the losses due to the disease. During the 1990s the disease was shown to be caused by multiple begomoviruses and a single, disease-specific betasatellite. Post-resistance breaking the complex encompassed only a single begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and a recombinant version of the betasatellite. Surprisingly CLCuBuV lacks an intact transcriptional-activator protein (TrAP) gene. The TrAP gene is found in all begomoviruses and encodes a product of ∼134 amino acids that is important in virus-host interactions; being a suppressor of post-transcriptional gene silencing (host defence) and a transcription factor that modulates host gene expression, including microRNA genes. Recent studies have highlighted the differences between CLCuBuV and the earlier viruses that are part of on-going efforts to define the molecular basis for resistance breaking in cotton. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Use of tissue expanders in the repair of complex abdominal wall defects.

    Science.gov (United States)

    Clifton, Matthew S; Heiss, Kurt F; Keating, Jane J; Mackay, Greg; Ricketts, Richard R

    2011-02-01

    Closure of abdominal wall defects in children poses a challenge for pediatric surgeons. We describe a technique using tissue expanders placed either intraperitoneally or in the abdominal wall to aid in the reconstruction of a variety of complex abdominal wall defects. The tissue expanders are inserted under general anesthesia. Initial expansion is done in the operating room with attention to peak airway pressure, urine output, and end-tidal carbon dioxide. The expanders are inflated in the outpatient setting via percutaneous access until the calculated inflation volume is achieved. They are then removed; and definitive closure is accomplished using a combination of native tissue flaps, abdominal component separation techniques, biomaterials, and synthetic material. Six children underwent tissue expansion for treatment of abdominal wall defects (omphalocele, n = 3), trauma (n = 1), and thoracopagus twins (n = 1 pair). One to 4 expanders were used per patient, with all having a successful reconstruction of their abdominal walls. Two to 3 operations were required to restore abdominal domain and consisted of expander insertion, removal with reconstruction, and possible revision of the reconstruction. Tissue expanders possess a broad range of applications for abdominal wall reconstruction and can be used in patients of all ages. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. The Cockayne syndrome B protein, involved in transcription-coupled repair resides in a RNA polymerase II-containing complex.

    NARCIS (Netherlands)

    A.J. van Gool (Alain); E. Citterio (Elisabetta); S. Rademakers (Suzanne); R. van Os; W. Vermeulen (Wim); A. Constantinou; J-M. Egly (Jean-Marc); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1997-01-01

    textabstractTranscription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked

  17. Repair of chronic rupture of the achilles tendon using 2 intratendinous flaps from the proximal gastrocnemius-soleus complex.

    Science.gov (United States)

    El Shewy, Mohamed Taha; El Barbary, Hassan Magdy; Abdel-Ghani, Hisham

    2009-08-01

    Chronic rupture of the Achilles tendon is a surgical challenge, owing to the presence of a gap between the retracted ends, which renders direct repair almost impossible. In this study, 2 intratendinous distally based flaps fashioned from the proximal gastrocnemiussoleus complex are used to bridge the gap between the retracted edges of the ruptured Achilles tendon. The flaps are placed in the same line of pull of the ruptured tendon, in an effort to make the graft mimic the original biomechanics as much as possible. Case series; Level of evidence, 4. Eleven patients (9 male and 2 female) with neglected ruptures of the Achilles tendon with retracted ends were included in this study. Two flaps fashioned from the proximal gastrocnemiussoleus complex were rotated over themselves, passed through the proximal stump, and then securely inserted into a previously prepared bed in the distal stump. The patients were followed up for a period of 6 to 9 years. At the final follow-up, all patients were able to return to their preinjury level of activity within a period of 6 to 9 months. The mean preoperative American Orthopedic Foot and Ankle Society score was 42.27, whereas it was 98.91 at the final follow-up, with a range of 88 (in 1 patient) to 100 points (in 10 patients). All 11 patients showed statistically significant improvement according to the Holz rating system. This technique allows for a bridging of the defect present in chronic ruptures of Achilles tendons, with a minimum of complications and a good final outcome.

  18. The DNA translocase RAD5A acts independently of the other main DNA repair pathways, and requires both its ATPase and RING domain for activity in Arabidopsis thaliana.

    Science.gov (United States)

    Klemm, Tobias; Mannuß, Anja; Kobbe, Daniela; Knoll, Alexander; Trapp, Oliver; Dorn, Annika; Puchta, Holger

    2017-08-01

    Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error-free branch of post-replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication-associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single-strand break repair (AtPARP1), as well as microhomology-mediated double-strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM-mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  19. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  20. Break induced replication in eukaryotes: mechanisms, functions, and consequences.

    Science.gov (United States)

    Sakofsky, Cynthia J; Malkova, Anna

    2017-08-01

    Break-induced replication (BIR) is an important pathway specializing in repair of one-ended double-strand DNA breaks (DSBs). This type of DSB break typically arises at collapsed replication forks or at eroded telomeres. BIR initiates by invasion of a broken DNA end into a homologous template followed by initiation of DNA synthesis that can proceed for hundreds of kilobases. This synthesis is drastically different from S-phase replication in that instead of a replication fork, BIR proceeds via a migrating bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual mode of DNA replication is responsible for frequent genetic instabilities associated with BIR, including hyper-mutagenesis, which can lead to the formation of mutation clusters, extensive loss of heterozygosity, chromosomal translocations, copy-number variations and complex genomic rearrangements. In addition to budding yeast experimental systems that were initially employed to investigate eukaryotic BIR, recent studies in different organisms including humans, have provided multiple examples of BIR initiated within different cellular contexts, including collapsed replication fork and telomere maintenance in the absence of telomerase. In addition, significant progress has been made towards understanding microhomology-mediated BIR (MMBIR) that can promote complex chromosomal rearrangements, including those associated with cancer and those leading to a number of neurological disorders in humans.

  1. Reliable complex abdominal wall hernia repairs with a narrow, well-fixed retrorectus polypropylene mesh: A review of over 100 consecutive cases.

    Science.gov (United States)

    Lanier, Steven T; Fligor, Jennifer E; Miller, Kyle R; Dumanian, Gregory A

    2016-12-01

    Our objective was to determine outcomes for complex ventral hernia repairs in a large cohort of patients utilizing an operative construct employing retrorectus placement of a narrow, macroporous polypropylene mesh with up to 45 suture fixation points for force distribution. No consensus exists on the optimal technique for repair of complex ventral hernias. Current trends emphasize large meshes with wide overlaps and minimal suture fixation, though reported complications and recurrence remain problematic. A retrospective review was performed for all patients undergoing ventral hernia repair with retrorectus placement of midweight, uncoated, soft polypropylene mesh by a single surgeon (GAD) between the years of 2010 and 2015. Patient characteristics, operative history, operative data, and postoperative course were reviewed. A total of 101 patients with a mean age of 56 years and a mean body mass index of 29 m/kg(2) (range 18-51 m/kg(2)) underwent hernia repair. Patients had a median of 3 prior abdominal operations (range 0-9), with 44 patients presenting with recurrent hernias. A total of 42 patients were Ventral Hernia Working Group grade 1, 40 grade 2, 17 grade 3, and 2 grade 4. There were no recurrences at a mean follow-up of almost 400 days for the 93 patients with long-term follow-up. The surgical site occurrence rate was 7.9% (3 surgical site infections, 2 seromas, 2 hematomas, and 4 instances of delayed wound healing in 8 patients). One patient required reoperation for hematoma drainage; 5 patients required readmission within 30 days. An operative construct employing a retrorectus placement of a narrow, macroporous polypropylene mesh with up to 45 suture fixation points for force distribution can achieve significantly better outcomes across a spectrum of Ventral Hernia Working Group grade, risk-stratified patients compared to rates reported in the literature for current strategies that employ wide meshes with minimal fixation. Copyright © 2016 Elsevier

  2. The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex.

    Science.gov (United States)

    Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E; Jaspers, Nicolaas G J; Kaptein, Robert; Hoeijmakers, Jan H J; Boelens, Rolf

    2015-08-14

    The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. DNA repair mechanisms and gametogenesis

    NARCIS (Netherlands)

    W.M. Baarends (Willy); R. van der Laan (Roald); J.A. Grootegoed (Anton)

    2001-01-01

    textabstractIn mammals, there is a complex and intriguing relationship between DNA repair and gametogenesis. DNA repair mechanisms are involved not only in the repair of different types of DNA damage in developing germline cells, but also take part in the meiotic

  4. Breaking Boundaries

    DEFF Research Database (Denmark)

    produce desperate attempts to maintain old or create new differences. Political and sociological research into these complex processes has been mainly guided by structural and normative concerns. Faced with growing evidence about the instability of world order and domestic social structures alike, policy....... As a fundamental human experience, liminality transmits cultural practices, codes, rituals, and meanings in-between aggregate structures and uncertain outcomes. As a methodological tool it is well placed to overcome disciplinary boundaries, which often direct attention to specific structures or sectors of society....... Its capacity to provide explanatory accounts of seemingly unstructured situations provides an opportunity to link experience-based and culture-oriented approaches not only to contemporary problems but also to undertake comparisons across historical periods. From a perspective of liminality...

  5. XRCC1 coordinates disparate responses and multiprotein repair complexes depending on the nature and context of the DNA damage

    DEFF Research Database (Denmark)

    Hanssen-Bauer, Audun; Solvang-Garten, Karin; Sundheim, Ottar

    2011-01-01

    short patch (SP) and long patch (LP) base excision repair (BER). We show that POLß and PNK colocalize with XRCC1 in replication foci and that POLß and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied....... We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLß and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP......-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLß to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER...

  6. Statistical Dependence of Pipe Breaks on Explanatory Variables

    Directory of Open Access Journals (Sweden)

    Patricia Gómez-Martínez

    2017-02-01

    Full Text Available Aging infrastructure is the main challenge currently faced by water suppliers. Estimation of assets lifetime requires reliable criteria to plan assets repair and renewal strategies. To do so, pipe break prediction is one of the most important inputs. This paper analyzes the statistical dependence of pipe breaks on explanatory variables, determining their optimal combination and quantifying their influence on failure prediction accuracy. A large set of registered data from Madrid water supply network, managed by Canal de Isabel II, has been filtered, classified and studied. Several statistical Bayesian models have been built and validated from the available information with a technique that combines reference periods of time as well as geographical location. Statistical models of increasing complexity are built from zero up to five explanatory variables following two approaches: a set of independent variables or a combination of two joint variables plus an additional number of independent variables. With the aim of finding the variable combination that provides the most accurate prediction, models are compared following an objective validation procedure based on the model skill to predict the number of pipe breaks in a large set of geographical locations. As expected, model performance improves as the number of explanatory variables increases. However, the rate of improvement is not constant. Performance metrics improve significantly up to three variables, but the tendency is softened for higher order models, especially in trunk mains where performance is reduced. Slight differences are found between trunk mains and distribution lines when selecting the most influent variables and models.

  7. BRD4 Promotes DNA Repair and Mediates the Formation of TMPRSS2-ERG Gene Rearrangements in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Xiangyi Li

    2018-01-01

    Full Text Available BRD4 belongs to the bromodomain and extraterminal (BET family of chromatin reader proteins that bind acetylated histones and regulate gene expression. Pharmacological inhibition of BRD4 by BET inhibitors (BETi has indicated antitumor activity against multiple cancer types. We show that BRD4 is essential for the repair of DNA double-strand breaks (DSBs and mediates the formation of oncogenic gene rearrangements by engaging the non-homologous end joining (NHEJ pathway. Mechanistically, genome-wide DNA breaks are associated with enhanced acetylation of histone H4, leading to BRD4 recruitment, and stable establishment of the DNA repair complex. In support of this, we also show that, in clinical tumor samples, BRD4 protein levels are negatively associated with outcome after prostate cancer (PCa radiation therapy. Thus, in addition to regulating gene expression, BRD4 is also a central player in the repair of DNA DSBs, with significant implications for cancer therapy.

  8. CRISPR Technology Reveals RAD(51)-ical Mechanisms of Repair in Roundworms: An Educational Primer for Use with "Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans".

    Science.gov (United States)

    Turcotte, Carolyn A; Andrews, Nicolas P; Sloat, Solomon A; Checchi, Paula M

    2016-11-01

    The mechanisms cells use to maintain genetic fidelity via DNA repair and the accuracy of these processes have garnered interest from scientists engaged in basic research to clinicians seeking improved treatment for cancer patients. Despite the continued advances, many details of DNA repair are still incompletely understood. In addition, the inherent complexity of DNA repair processes, even at the most fundamental level, makes it a challenging topic. This primer is meant to assist both educators and students in using a recent paper, "Promotion of homologous recombination by SWS-1 in complex with RAD-51 paralogs in Caenorhabditis elegans," to understand mechanisms of DNA repair. The goals of this primer are to highlight and clarify several key techniques utilized, with special emphasis on the clustered, regularly interspaced, short palindromic repeats technique and the ways in which it has revolutionized genetics research, as well as to provide questions for deeper in-class discussion. Copyright © 2016 by the Genetics Society of America.

  9. Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies.

    Directory of Open Access Journals (Sweden)

    Cristina Rivellini

    Full Text Available Remodeling of extracellular matrix (ECM is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.

  10. Code breaking in the pacific

    CERN Document Server

    Donovan, Peter

    2014-01-01

    Covers the historical context and the evolution of the technically complex Allied Signals Intelligence (Sigint) activity against Japan from 1920 to 1945 Describes, explains and analyzes the code breaking techniques developed during the war in the Pacific Exposes the blunders (in code construction and use) made by the Japanese Navy that led to significant US Naval victories

  11. Stripped-down DNA repair in a highly reduced parasite

    Directory of Open Access Journals (Sweden)

    Fast Naomi M

    2007-03-01

    Full Text Available Abstract Background Encephalitozoon cuniculi is a member of a distinctive group of single-celled parasitic eukaryotes called microsporidia, which are closely related to fungi. Some of these organisms, including E. cuniculi, also have uniquely small genomes that are within the prokaryotic range. Thus, E. cuniculi has undergone a massive genome reduction which has resulted in a loss of genes from diverse biological pathways, including those that act in DNA repair. DNA repair is essential to any living cell. A loss of these mechanisms invariably results in accumulation of mutations and/or cell death. Six major pathways of DNA repair in eukaryotes include: non-homologous end joining (NHEJ, homologous recombination repair (HRR, mismatch repair (MMR, nucleotide excision repair (NER, base excision repair (BER and methyltransferase repair. DNA polymerases are also critical players in DNA repair processes. Given the close relationship between microsporidia and fungi, the repair mechanisms present in E. cuniculi were compared to those of the yeast Saccharomyces cerevisiae to ascertain how the process of genome reduction has affected the DNA repair pathways. Results E. cuniculi lacks 16 (plus another 6 potential absences of the 56 DNA repair genes sought via BLASTP and PSI-BLAST searches. Six of 14 DNA polymerases or polymerase subunits are also absent in E. cuniculi. All of these genes are relatively well conserved within eukaryotes. The absence of genes is not distributed equally among the different repair pathways; some pathways lack only one protein, while there is a striking absence of many proteins that are components of both double strand break repair pathways. All specialized repair polymerases are also absent. Conclusion Given the large number of DNA repair genes that are absent from the double strand break repair pathways, E. cuniculi is a prime candidate for the study of double strand break repair with minimal machinery. Strikingly, all of the

  12. Complex aortic and bilateral renal artery aneurysm repair in a young patient with multiple arterial aneurysm syndrome

    Directory of Open Access Journals (Sweden)

    Carlos A. Hinojosa, MD, MSc

    2016-09-01

    Full Text Available Aneurysmal disease affecting the aorta and visceral vessels in young patients is uncommon and typically associated with connective tissue disorders. We describe the case of a 17-year-old girl who presented with acute onset of abdominal pain; computed tomography scan revealed aortic and bilateral renal artery aneurysms and a perirenal hematoma. She was taken to the angiography suite; rupture of the right renal artery aneurysm was identified and immediately treated successfully with coil embolization. The left renal artery aneurysm was repaired with ex-vivo renal autotransplantation; 2 years later, the aorta and right renal artery underwent surgical reconstruction.

  13. Tricuspid valve mycetoma in an infant successfully treated by excision and complex tricuspid valve repair followed by fluconazole therapy.

    Science.gov (United States)

    Anil Kumar, V; Francis, Edwin; Sreehari, Sreekala; Raj, Benedict

    2014-04-01

    Fungal valve endocarditis in children is an uncommon and lethal disease. The risk increases with use of central venous catheters (CVC), total parenteral nutrition (TPN), and use of broad-spectrum antibiotics during the neonatal period. Due to high mortality, a combination of surgery and antifungal therapy is usually recommended for treatment. Case report and review of the literature. We present a case of an asymptomatic infant with multiple Candida tricuspid valve mycetomas. Complete cure was achieved by combined tricuspid valve repair and fluconazole therapy. We also review 26 cases of tricuspid valve Candida endocarditis in children published in the literature. From being uniformly fatal five decades ago to a current survival rate of 64% to 100%, the prognosis of Candida endocarditis has changed dramatically with the use of antifungal therapy alone or in combination with surgery. Our case re-emphasizes the role of valve-sparing debridement with repair of the native valve using autologous pericardium in combination with long-term antifungal therapy as a feasible option in managing tricuspid valve Candida endocarditis.

  14. Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II.

    Science.gov (United States)

    Boehm, M; Yu, J; Reisinger, V; Beckova, M; Eichacker, L A; Schlodder, E; Komenda, J; Nixon, P J

    2012-12-19

    Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680(+), was similar to that of PSII complexes lacking the Mn(4)CaO(5) cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.

  15. 46 CFR 196.30-20 - Breaking of safety valve seal.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Breaking of safety valve seal. 196.30-20 Section 196.30... OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-20 Breaking of safety valve seal. (a) If at any time it is necessary to break the seal on a safety valve for any purpose, the Chief...

  16. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. [Los Alamos National Lab., NM (United States); Chen, D.S. [Rochester Univ., NY (United States). Dept. of Radiation Oncology

    1993-02-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  17. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. (Los Alamos National Lab., NM (United States)); Chen, D.S. (Rochester Univ., NY (United States). Dept. of Radiation Oncology)

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  18. Hypospadias repair

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003000.htm Hypospadias repair To use the sharing features on this page, please enable JavaScript. Hypospadias repair is surgery to correct a defect in ...

  19. Bioaccumulation and cancer risk of polycyclic aromatic hydrocarbons in leafy vegetables grown in soils within automobile repair complex and environ in Uyo, Nigeria.

    Science.gov (United States)

    Inam, Edu; Ibanga, Felicia; Essien, Joseph

    2016-12-01

    Using gas chromatography-mass spectrometry and an incremental lifetime cancer risks (ILCRs) assessment model, the bioaccumulation and cancer risk of 16 USEPA priority polycyclic aromatic hydrocarbons (PAHs) in leafy vegetables (Vernonia amygdalina and Lasianthera africanum) grown in soils within an automobile repair complex environment in Uyo, Nigeria was studied. The total PAHs concentrations recorded for soils ranged from 0.02 to 1.77 mg/kg. The highest level of 1.77 mg/kg was recorded for soils from the main automobile repair complex (site 1). Low molecular weight (LMW) PAHs were predominant although some high molecular weight (HMW) PAHs suites (0.04 mg/kg of chrysene and 0.04 of benzo[k]fluoranthene) were also found in site 1. The leafy vegetables accumulated PAHs were mostly LMW. Accumulation levels were similar but the extent of PAH uptake in vegetables was species dependent as V. amygdalina accumulated more (0.81 mg/kg). The bioaccumulation factors (BaFs) calculated ranged from 0.22 to 0.63 for L. africanum, and 0.18 to 0.55 for V. amygdalina in site 1 where high PAH levels were recorded in soil. Pearson correlation coefficient analysis revealed a strong positive relation between the PAH content of soil and the amount accumulated by L. africanum (r = 0.5) and V. amygdalina (r = 0.8) at p = 0.05. The vegetable's potential to bioaccumulate PAHs is indicative of their use as good bioindicators for PAH contamination in soil. Only two of the USEPA possible human carcinogenic PAHs were detected, and carcinogenic risk assessment based on occupational exposures to soil particles by adults revealed that the total risk level (7.17 × 10-5) contribution from incidental soil ingestion, dermal contact, and soil particle dust inhalation slightly exceed the USEPA acceptable limits (vegetables grown in and around automobile repair complexes across Nigeria.

  20. An Injectable Complex of β-tricalcium Phosphate Granules, Hyaluronate, and rhFGF-2 on Repair of Long-bone Fractures with Large Fragments

    Science.gov (United States)

    Tanaka, T; Kumagae, Y; Chazono, M; Komaki, H; Kitasato, S; Kakuta, A; Marumo, K

    2014-01-01

    We evaluated the effects of an injectable complex of β-tricalcium phosphate (β-TCP) granules, hyaluronate, and recombinant human fibroblast growth factor-2 (rhFGF-2) on repair of unstable intertrochanteric fractures in elderly patients. Twenty-five patients (range, 76-91 years) having 31.A2 fractures (AO classification) were treated with injection of the complex followed by intramedullary nails. Bone regeneration and β-TCP resorption, unions of intertrochanteric fractures and displaced lesser trochanters to the shaft, and varus deformity of the femoral neck were assessed by X-ray and CT scans. Fracture union occurred in all cases and union of the displaced lesser trochanter to the shaft was obtained in 24 cases by 12 weeks. It is of interest that β-TCP granules were completely resorbed and marked new bone formation around the lesser trochanter was observed in all cases compared to cases not treated with the complex. Based on the results of intertrochanteric fractures, we applied this technique to two patients with subtrochanteric or humeral fractures in elderly patients, and obtained bone union. This complex is a paste-like material that is easy to handle, and it may be of considerable use in treatment of both unstable intertrochanteric fractures and other cortical bone defects with minimal surgical invasion. PMID:25246986

  1. An Injectable Complex of β-tricalcium Phosphate Granules, Hyaluronate, and rhFGF-2 on Repair of Long-bone Fractures with Large Fragments.

    Science.gov (United States)

    Tanaka, T; Kumagae, Y; Chazono, M; Komaki, H; Kitasato, S; Kakuta, A; Marumo, K

    2014-01-01

    We evaluated the effects of an injectable complex of β-tricalcium phosphate (β-TCP) granules, hyaluronate, and recombinant human fibroblast growth factor-2 (rhFGF-2) on repair of unstable intertrochanteric fractures in elderly patients. Twenty-five patients (range, 76-91 years) having 31.A2 fractures (AO classification) were treated with injection of the complex followed by intramedullary nails. Bone regeneration and β-TCP resorption, unions of intertrochanteric fractures and displaced lesser trochanters to the shaft, and varus deformity of the femoral neck were assessed by X-ray and CT scans. Fracture union occurred in all cases and union of the displaced lesser trochanter to the shaft was obtained in 24 cases by 12 weeks. It is of interest that β-TCP granules were completely resorbed and marked new bone formation around the lesser trochanter was observed in all cases compared to cases not treated with the complex. Based on the results of intertrochanteric fractures, we applied this technique to two patients with subtrochanteric or humeral fractures in elderly patients, and obtained bone union. This complex is a paste-like material that is easy to handle, and it may be of considerable use in treatment of both unstable intertrochanteric fractures and other cortical bone defects with minimal surgical invasion.

  2. Application of computer-aided three-dimensional skull model with rapid prototyping technique in repair of zygomatico-orbito-maxillary complex fracture.

    Science.gov (United States)

    Li, Wei Zhong; Zhang, Mei Chao; Li, Shao Ping; Zhang, Lei Tao; Huang, Yu

    2009-06-01

    With the advent of CAD/CAM and rapid prototyping (RP), a technical revolution in oral and maxillofacial trauma was promoted to benefit treatment, repair of maxillofacial fractures and reconstruction of maxillofacial defects. For a patient with zygomatico-facial collapse deformity resulting from a zygomatico-orbito-maxillary complex (ZOMC) fracture, CT scan data were processed by using Mimics 10.0 for three-dimensional (3D) reconstruction. The reduction design was aided by 3D virtual imaging and the 3D skull model was reproduced using the RP technique. In line with the design by Mimics, presurgery was performed on the 3D skull model and the semi-coronal incision was taken for reduction of ZOMC fracture, based on the outcome from the presurgery. Postoperative CT and images revealed significantly modified zygomatic collapse and zygomatic arch rise and well-modified facial symmetry. The CAD/CAM and RP technique is a relatively useful tool that can assist surgeons with reconstruction of the maxillofacial skeleton, especially in repairs of ZOMC fracture.

  3. H4K20me0 marks post-replicative chromatin and recruits the TONSL₋MMS22L DNA repair complex

    Energy Technology Data Exchange (ETDEWEB)

    Saredi, Giulia; Huang, Hongda; Hammond, Colin M.; Alabert, Constance; Bekker-Jensen, Simon; Forne, Ignasi; Reverón-Gómez, Nazaret; Foster, Benjamin M.; Mlejnkova, Lucie; Bartke, Till; Cejka, Petr; Mailand, Niels; Imhof, Axel; Patel, Dinshaw J.; Groth, Anja [UCopenhagen; (MSKCC); (ICL); (LMU); (Zurich)

    2016-06-22

    Here, we report that after DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. In this paper we reveal that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L1, 2, 3, 4 homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new histones incorporated during DNA replication and mark post-replicative chromatin until the G2/M phase of the cell cycle. Accordingly, TONSL–MMS22L binds new histones H3–H4 both before and after incorporation into nucleosomes, remaining on replicated chromatin until late G2/M. H4K20me0 recognition is required for TONSL–MMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Finally, together, these data reveal a histone-reader-based mechanism for recognizing the post-replicative state, offering a new angle to understand DNA repair with the potential for targeted cancer therapy.

  4. Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

    Science.gov (United States)

    Wu, Yuliang; Sommers, Joshua A.; Suhasini, Avvaru N.; Leonard, Thomas; Deakyne, Julianna S.; Mazin, Alexander V.; Shin-ya, Kazuo; Kitao, Hiroyuki

    2010-01-01

    Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair. PMID:20639400

  5. Network repair based on community structure

    Science.gov (United States)

    Wang, Tianyu; Zhang, Jun; Sun, Xiaoqian; Wandelt, Sebastian

    2017-06-01

    Real-world complex systems are often fragile under disruptions. Accordingly, research on network repair has been studied intensively. Recently proposed efficient strategies for network disruption, based on collective influence, call for more research on efficient network repair strategies. Existing strategies are often designed to repair networks with local information only. However, the absence of global information impedes the creation of efficient repairs. Motivated by this limitation, we propose a concept of community-level repair, which leverages the community structure of the network during the repair process. Moreover, we devise a general framework of network repair, with in total six instances. Evaluations on real-world and random networks show the effectiveness and efficiency of the community-level repair approaches, compared to local and random repairs. Our study contributes to a better understanding of repair processes, and reveals that exploitation of the community structure improves the repair process on a disrupted network significantly.

  6. Srs2: the "Odd-Job Man" in DNA repair.

    Science.gov (United States)

    Marini, Victoria; Krejci, Lumir

    2010-03-02

    Homologous recombination plays a key role in the maintenance of genome integrity, especially during DNA replication and the repair of double-stranded DNA breaks (DSBs). Just a single un-repaired break can lead to aneuploidy, genetic aberrations or cell death. DSBs are caused by a vast number of both endogenous and exogenous agents including genotoxic chemicals or ionizing radiation, as well as through replication of a damaged template DNA or the replication fork collapse. It is essential for cell survival to recognise and process DSBs as well as other toxic intermediates and launch most appropriate repair mechanism. Many helicases have been implicated to play role in these processes, however their detail roles, specificities and co-operativity in the complex protein-protein interaction networks remain unclear. In this review we summarize the current knowledge about Saccharomyces cerevisiae helicase Srs2 and its effect on multiple DNA metabolic processes that generally affect genome stability. It would appear that Srs2 functions as an "Odd-Job Man" in these processes to make sure that the jobs proceed when and where they are needed. (c) 2010 Elsevier B.V. All rights reserved.

  7. H4K20me0 marks post-replicative chromatin and recruits the TONSL–MMS22L DNA repair complex

    DEFF Research Database (Denmark)

    Saredi, Giulia; Huang, Hongda; Hammond, Colin M

    2016-01-01

    is required for TONSL–MMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Together, these data reveal a histone-reader-based mechanism......After DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. Here we reveal...... that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific...

  8. DNA Repair Deficiency in Neurodegeneration

    Science.gov (United States)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A.; Stevnsner, Tinna

    2011-01-01

    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 base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby causing Huntington’s disease. Single-strand breaks are common DNA lesions and are associated with the neurodegenerative diseases, ataxia-oculomotor apraxia-1 and spinocerebellar ataxia with axonal neuropathy-1. DNA double-strand breaks are toxic lesions and two main pathways exist for their repair: 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 may be linked with the age-associated neurodegenerative disorders Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Mutation in the WRN protein leads to the premature aging disease Werner syndrome, a disorder that features neurodegeneration. In this article we review the evidence linking deficiencies in the DNA repair pathways with neurodegeneration. PMID:21550379

  9. Tribute to dr louis keith: twin and physician extraordinaire/twin research reports: influences on asthma severity; chimerism revisited; DNA strand break repair/media reports: twins born apart; elevated twin frequencies; celebrity father of twins; conjoined twinning.

    Science.gov (United States)

    Segal, Nancy L

    2014-10-01

    The International Society for Twin Studies has lost a valued friend and colleague. Dr Louis Keith, Emeritus Professor of Obstetrics and Gynecology at Northwestern University, in Chicago, passed away on Sunday, July 6, 2014. His life and work with twins will be acknowledged at the November 2014 International Twin Congress in Budapest, Hungary. Next, twin research reports on the severity of asthma symptoms, a case of chimerism, and factors affecting DNA breakage and repair mechanisms are reviewed. Media reports cover twins born apart, elevated twin frequencies, a celebrity father of twins, and a family's decision to keep conjoined twins together.

  10. Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation

    Energy Technology Data Exchange (ETDEWEB)

    Asaithamby, Aroumougame, E-mail: Aroumougame.Asaithamy@UTsouthwestern.edu [Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 (United States); Chen, David J., E-mail: David.Chen@UTsouthwestern.edu [Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 (United States)

    2011-06-03

    Low-linear energy transfer (LET) radiation (i.e., {gamma}- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles is slowly repaired or is irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure.

  11. Evidence for a repair enzyme complex involving ERCC1, and the correcting activities of ERCC4, ERCC11 and the xeroderma pigmentosum group F.

    NARCIS (Netherlands)

    A.J. van Vuuren (Hanneke); E. Appeldoorn (Esther); H. Odijk (Hanny); A. Yasui (Akira); N.G.J. Jaspers (Nicolaas); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractNucleotide excision repair (NER), one of the major cellular DNA repair systems, removes a wide range of lesions in a multi-enzyme reaction. In man, a NER defect due to a mutation in one of at least 11 distinct genes, can give rise to the inherited repair disorders xeroderma pigmentosum

  12. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair

    DEFF Research Database (Denmark)

    Kanu, N.; Grönroos, E.; Martinez, P.

    2015-01-01

    proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal......-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication......, suppression of replication stress and the coordination of DNA repair....

  13. Bladder exstrophy repair

    Science.gov (United States)

    Bladder birth defect repair; Everted bladder repair; Exposed bladder repair; Repair of bladder exstrophy ... Bladder exstrophy repair involves two surgeries. The first surgery is to repair the bladder. The second one ...

  14. Recognition and Repair of Communicative Failures: The Interaction between Theory of Mind and Cognitive Complexity in Schizophrenic Patients

    Science.gov (United States)

    Bosco, Francesca M.; Bono, Adele; Bara, Bruno G.

    2012-01-01

    The aim of the present research is to perform a detailed and empirical investigation of schizophrenia patients' deficits in recognizing and recovering a communicative failure. In particular, this paper investigates the role of Theory of Mind (ToM) and of the complexity of the mental representations involved in explaining patients' deficits in…

  15. Clubfoot repair

    Science.gov (United States)

    ... Clubfoot release; Talipes equinovarus - repair; Tibialis anterior tendon transfer Patient ... of the foot. In: Herring JA, ed. Tachdjian's Pediatric Orthopaedics . 5th ed. Philadelphia, PA: Elsevier Saunders; 2014: ...

  16. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2011-01-01

    causing Huntington's disease. Single-strand breaks are common DNA lesions and are associated with the neurodegenerative diseases, ataxia-oculomotor apraxia-1 and spinocerebellar ataxia with axonal neuropathy-1. DNA double-strand breaks are toxic lesions and two main pathways exist for their repair......: 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...

  17. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten

    2010-01-01

    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  18. The Association of Low-Penetrance Variants in DNA Repair Genes with Colorectal Cancer: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Aggarwal, Nikhil; Donald, Neil D; Malik, Salim; Selvendran, Subothini S; McPhail, Mark Jw; Monahan, Kevin J

    2017-07-27

    Approximately 35% of colorectal cancer (CRC) risk is attributable to heritable factors known hereditary syndromes, accounting for 6%. The remainder may be due to lower penetrance polymorphisms particularly of DNA repair genes. DNA repair pathways, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), direct reversal repair (DRR), and double-strand break repair are complex, evolutionarily conserved, and critical in carcinogenesis. Germline mutations in these genes are associated with high-penetrance CRC syndromes such as Lynch syndrome. However, the association of low-penetrance polymorphisms of DNA repair genes with CRC risk remains unclear. A systematic literature review of PubMed, Embase, and HuGENet databases was conducted. Pre-specified criteria determined study inclusion/exclusion. Per-allele, pooled odds ratios disclosed the risk attributed to each variant. Heterogeneity was investigated by subgroup analyses for ethnicity and tumor location; funnel plots and Egger's test assessed publication bias. Sixty-one polymorphisms in 26 different DNA repair genes were identified. Meta-analyses for 22 polymorphisms in 17 genes revealed that six polymorphisms were significantly associated with CRC risk within BER (APE1, PARP1), NER (ERCC5, XPC), double-strand break (RAD18), and DRR (MGMT), but none within MMR. Subgroup analyses revealed significant association of OGG1 rs1052133 with rectal cancer risk. Egger's test revealed no publication bias. Low-penetrance polymorphisms in DNA repair genes alter susceptibility to CRC. Future studies should therefore analyze whole-genome polymorphisms and any synergistic effects on CRC risk.Translational impact:This knowledge may enhance CRC risk assessment and facilitate a more personalized approach to cancer prevention.

  19. DNA-Protein Crosslink Proteolysis Repair.

    Science.gov (United States)

    Vaz, Bruno; Popovic, Marta; Ramadan, Kristijan

    2017-06-01

    Proteins that are covalently bound to DNA constitute a specific type of DNA lesion known as DNA-protein crosslinks (DPCs). DPCs represent physical obstacles to the progression of DNA replication. If not repaired, DPCs cause stalling of DNA replication forks that consequently leads to DNA double-strand breaks, the most cytotoxic DNA lesion. Although DPCs are common DNA lesions, the mechanism of DPC repair was unclear until now. Recent work unveiled that DPC repair is orchestrated by proteolysis performed by two distinct metalloproteases, SPARTAN in metazoans and Wss1 in yeast. This review summarizes recent discoveries on two proteases in DNA replication-coupled DPC repair and establishes DPC proteolysis repair as a separate DNA repair pathway for genome stability and protection from accelerated aging and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Homeostatic nuclear RAGE–ATM interaction is essential for efficient DNA repair

    Science.gov (United States)

    Kumar, Varun; Fleming, Thomas; Terjung, Stefan; Gorzelanny, Christian; Gebhardt, Christoffer; Agrawal, Raman; Mall, Marcus A.; Ranzinger, Julia; Zeier, Martin; Madhusudhan, Thati; Ranjan, Satish; Isermann, Berend; Liesz, Arthur; Deshpande, Divija; Häring, Hans-Ulrich; Biswas, Subrata K; Reynolds, Paul R.; Hammes, Hans-Peter; Peperkok, Rainer; Angel, Peter; Herzig, Stephan

    2017-01-01

    Abstract The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE−/−), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis. PMID:28977635

  1. Kinetic theory approach to modeling of cellular repair mechanisms under genome stress.

    Directory of Open Access Journals (Sweden)

    Jinpeng Qi

    Full Text Available Under acute perturbations from outer environment, a normal cell can trigger cellular self-defense mechanism in response to genome stress. To investigate the kinetics of cellular self-repair process at single cell level further, a model of DNA damage generating and repair is proposed under acute Ion Radiation (IR by using mathematical framework of kinetic theory of active particles (KTAP. Firstly, we focus on illustrating the profile of Cellular Repair System (CRS instituted by two sub-populations, each of which is made up of the active particles with different discrete states. Then, we implement the mathematical framework of cellular self-repair mechanism, and illustrate the dynamic processes of Double Strand Breaks (DSBs and Repair Protein (RP generating, DSB-protein complexes (DSBCs synthesizing, and toxins accumulating. Finally, we roughly analyze the capability of cellular self-repair mechanism, cellular activity of transferring DNA damage, and genome stability, especially the different fates of a certain cell before and after the time thresholds of IR perturbations that a cell can tolerate maximally under different IR perturbation circumstances.

  2. A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia.

    Directory of Open Access Journals (Sweden)

    Mikołaj Słabicki

    Full Text Available DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP. We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

  3. Anti-tumour compounds illudin S and Irofulven induce DNA lesions ignored by global repair and exclusively processed by transcription- and replication-coupled repair pathways.

    Science.gov (United States)

    Jaspers, Nicolaas G J; Raams, Anja; Kelner, Michael J; Ng, Jessica M Y; Yamashita, Yukiko M; Takeda, Shiunichi; McMorris, Trevor C; Hoeijmakers, Jan H J

    2002-12-05

    Illudin S is a natural sesquiterpene drug with strong anti-tumour activity. Inside cells, unstable active metabolites of illudin cause the formation of as yet poorly characterised DNA lesions. In order to identify factors involved in their repair, we have performed a detailed genetic survey of repair-defective mutants for responses to the drug. We show that 90% of illudin's lethal effects in human fibroblasts can be prevented by an active nucleotide excision repair (NER) system. Core NER enzymes XPA, XPF, XPG, and TFIIH are essential for recovery. However, the presence of global NER initiators XPC, HR23A/HR23B and XPE is not required, whereas survival, repair and recovery from transcription inhibition critically depend on CSA, CSB and UVS, the factors specific for transcription-coupled NER. Base excision repair and non-homologous end-joining of DNA breaks do not play a major role in the processing of illudin lesions. However, active RAD18 is required for optimal cell survival, indicating that the lesions also block replication forks, eliciting post-replication-repair-like responses. However, the translesion-polymerase DNA pol eta is not involved. We conclude that illudin-induced lesions are exceptional in that they appear to be ignored by all of the known global repair systems, and can only be repaired when trapped in stalled replication or transcription complexes. We show that the semisynthetic illudin derivative hydroxymethylacylfulvene (HMAF, Irofulven), currently under clinical trial for anti-tumour therapy, acts via the same mechanism. Copyright 2002 Elsevier Science B.V.

  4. Variation in Base Excision Repair Capacity

    OpenAIRE

    Wilson, David M.; Kim, Daemyung; Berquist, Brian R.; Sigurdson, Alice J.

    2010-01-01

    The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence...

  5. Consistency of Trend Break Point Estimator with Underspecified Break Number

    Directory of Open Access Journals (Sweden)

    Jingjing Yang

    2017-01-01

    Full Text Available This paper discusses the consistency of trend break point estimators when the number of breaks is underspecified. The consistency of break point estimators in a simple location model with level shifts has been well documented by researchers under various settings, including extensions such as allowing a time trend in the model. Despite the consistency of break point estimators of level shifts, there are few papers on the consistency of trend shift break point estimators in the presence of an underspecified break number. The simulation study and asymptotic analysis in this paper show that the trend shift break point estimator does not converge to the true break points when the break number is underspecified. In the case of two trend shifts, the inconsistency problem worsens if the magnitudes of the breaks are similar and the breaks are either both positive or both negative. The limiting distribution for the trend break point estimator is developed and closely approximates the finite sample performance.

  6. EMC10 (Endoplasmic Reticulum Membrane Protein Complex Subunit 10) Is a Bone Marrow-Derived Angiogenic Growth Factor Promoting Tissue Repair After Myocardial Infarction.

    Science.gov (United States)

    Reboll, Marc R; Korf-Klingebiel, Mortimer; Klede, Stefanie; Polten, Felix; Brinkmann, Eva; Reimann, Ines; Schönfeld, Hans-Joachim; Bobadilla, Maria; Faix, Jan; Kensah, George; Gruh, Ina; Klintschar, Michael; Gaestel, Matthias; Niessen, Hans W; Pich, Andreas; Bauersachs, Johann; Gogos, Joseph A; Wang, Yong; Wollert, Kai C

    2017-11-07

    Clinical trials of bone marrow cell-based therapies after acute myocardial infarction (MI) have produced mostly neutral results. Treatment with specific bone marrow cell-derived secreted proteins may provide an alternative biological approach to improving tissue repair and heart function after MI. We recently performed a bioinformatic secretome analysis in bone marrow cells from patients with acute MI and discovered a poorly characterized secreted protein, EMC10 (endoplasmic reticulum membrane protein complex subunit 10), showing activity in an angiogenic screen. We investigated the angiogenic potential of EMC10 and its mouse homolog (Emc10) in cultured endothelial cells and infarcted heart explants. We defined the cellular sources and function of Emc10 after MI using wild-type, Emc10 -deficient, and Emc10 bone marrow-chimeric mice subjected to transient coronary artery ligation. Furthermore, we explored the therapeutic potential of recombinant Emc10 delivered by osmotic minipumps after MI in heart failure-prone FVB/N mice. Emc10 signaled through small GTPases, p21-activated kinase, and the p38 mitogen-activated protein kinase (MAPK)-MAPK-activated protein kinase 2 (MK2) pathway to promote actin polymerization and endothelial cell migration. Confirming the importance of these signaling events in the context of acute MI, Emc10 stimulated endothelial cell outgrowth from infarcted mouse heart explants via p38 MAPK-MK2. Emc10 protein abundance was increased in the infarcted region of the left ventricle and in the circulation of wild-type mice after MI. Emc10 expression was also increased in left ventricular tissue samples from patients with acute MI. Bone marrow-derived monocytes and macrophages were the predominant sources of Emc10 in the infarcted murine heart. Emc10 KO mice showed no cardiovascular phenotype at baseline. After MI, however, capillarization of the infarct border zone was impaired in KO mice, and the animals developed larger infarct scars and more

  7. Break-induced replication is highly inaccurate.

    Directory of Open Access Journals (Sweden)

    Angela Deem

    2011-02-01

    Full Text Available DNA must be synthesized for purposes of genome duplication and DNA repair. While the former is a highly accurate process, short-patch synthesis associated with repair of DNA damage is often error-prone. Break-induced replication (BIR is a unique cellular process that mimics normal DNA replication in its processivity, rate, and capacity to duplicate hundreds of kilobases, but is initiated at double-strand breaks (DSBs rather than at replication origins. Here we employed a series of frameshift reporters to measure mutagenesis associated with BIR in Saccharomyces cerevisiae. We demonstrate that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2,800-fold higher than during normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. We established that polymerase proofreading and mismatch repair correct BIR errors. Also, dNTP levels were elevated during BIR, and this contributed to BIR-related mutagenesis. We propose that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR, with Pol δ generating many of the mutagenic errors. We further postulate that activation of BIR in eukaryotic cells may significantly contribute to accumulation of mutations that fuel cancer and evolution.

  8. Genomic approaches to DNA repair and mutagenesis.

    Science.gov (United States)

    Wyrick, John J; Roberts, Steven A

    2015-12-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  10. Exonuclease 1 and its versatile roles in DNA repair

    DEFF Research Database (Denmark)

    Keijzers, Guido; Liu, Dekang; Rasmussen, Lene Juel

    2016-01-01

    Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin...

  11. Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

    Directory of Open Access Journals (Sweden)

    Rihito Morita

    2010-01-01

    Full Text Available DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.

  12. The predicted truncation from a cancer-associated variant of the MSH2 initiation codon alters activity of the MSH2-MSH6 mismatch repair complex

    OpenAIRE

    Cyr, Jennifer L.; Brown, Graham D; Stroop, Jennifer; Heinen, Christopher D.

    2011-01-01

    Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes. M M R recognizes and repairs DNA mismatches and small insertion/deletion loops. Carriers of MMR gene variants have a high risk of developing colorectal, endometrial, ovarian, and other extracolonic carcinomas. We report on an ovarian cancer patient who carries a germline MSH2 c.1A>C variant which alters the translation initiation codon. Mutations affecting the MSH2 start codon have been described previousl...

  13. Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair.

    Science.gov (United States)

    Gong, Fade; Clouaire, Thomas; Aguirrebengoa, Marion; Legube, Gaëlle; Miller, Kyle M

    2017-07-03

    Upon DNA damage, histone modifications are dynamically reshaped to accommodate DNA damage signaling and repair within chromatin. In this study, we report the identification of the histone demethylase KDM5A as a key regulator of the bromodomain protein ZMYND8 and NuRD (nucleosome remodeling and histone deacetylation) complex in the DNA damage response. We observe KDM5A-dependent H3K4me3 demethylation within chromatin near DNA double-strand break (DSB) sites. Mechanistically, demethylation of H3K4me3 is required for ZMYND8-NuRD binding to chromatin and recruitment to DNA damage. Functionally, KDM5A deficiency results in impaired transcriptional silencing and repair of DSBs by homologous recombination. Thus, this study identifies a crucial function for KDM5A in demethylating H3K4 to allow ZMYND8-NuRD to operate within damaged chromatin to repair DSBs. © 2017 Gong et al.

  14. Signalization and repair of the DNA double-strand breaks of in the cerebral tumors: modulation of the radiation response with the chemotherapy treatments; Signalization et reparation des cassures double-brin de l'ADN dans les gliomes: modulation de la reponse aux traitements chimio-radiotherapeutiques

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkova-Bencokova, Z

    2007-07-15

    There are about 6000 new cases of nervous system tumours each year in France. However, the current radio chemotherapeutic approaches against brain tumours remain still insufficient to produce a satisfactory therapeutic index. In parallel, the knowledge of the early radiobiological events has considerably progressed in the last few years. This thesis aims to provide new insights in the molecular and cellular response of brain tumours to radio chemotherapy. This thesis was divided into four stages. Stage 1: a novel DNA double-strand breaks repair pathway depending on the MRE11 protein but independent of the phosphorylation of H2AX emerged from the study of artefacts of the immunofluorescence technique and a systematic analysis of the radiosensitivity of human cells. Stage 2: the radiobiological features of 3 rodent models of glioma among the most used in preclinical trials and of 7 human glioma cell lines were investigated. Functional impairments of the BRCA1 protein in response to radiation and/or cisplatin were observed in the majority of the models tested, raising the question of the role of this protein in the anti-glioma treatments and in glioma genesis. Stage 3: in order to extend our approach to genetic syndromes associated with cerebral tumours predisposition, the radiobiological characteristics of the fibroblasts resulting from patients suffering from neurofibromatosis type 1 (NF1), a pathology associated with a strong incidence of peripheral nervous system tumours, were investigated. NF1 appeared to be a syndrome with moderated radiosensitivity, associated with a weak deficiency of DNA end-joining repair but with a strong activity of MRE11. These results enabled us to propose a preliminary model involving both proteins BRCA1 and NF1. Stage 4: considering the role of BRCA1 in the inhibition of some tyrosine kinase activity and in the response to cisplatin, we tested the radiobiological effects of treatments combining radiation, cisplatin and tyrosine kinase

  15. Repair process and a repaired component

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, III, Herbert Chidsey; Simpson, Stanley F.

    2018-02-20

    Matrix composite component repair processes are disclosed. The matrix composite repair process includes applying a repair material to a matrix composite component, securing the repair material to the matrix composite component with an external securing mechanism and curing the repair material to bond the repair material to the matrix composite component during the securing by the external securing mechanism. The matrix composite component is selected from the group consisting of a ceramic matrix composite, a polymer matrix composite, and a metal matrix composite. In another embodiment, the repair process includes applying a partially-cured repair material to a matrix composite component, and curing the repair material to bond the repair material to the matrix composite component, an external securing mechanism securing the repair material throughout a curing period, In another embodiment, the external securing mechanism is consumed or decomposed during the repair process.

  16. [Tricaicium phosphate complex pre-loaded with bone morphogenetic protein-2 or platelet derived growth factor-BB for repairing critical-size cranial defects in SD rats].

    Science.gov (United States)

    He, Rui-Xuan; Xiao, Jian-Bin; Song, Bing; Huang, Zhi-Hui; Zhao, Liang

    2016-03-01

    To observe the effect of a new biomaterial in promoting the bone regeneration for repairing critical-size cranial defects in SD rats. Critical-size cranial defects were induced in 3-month-old male Sprague-Dawley rats and repaired with the implants of calcium phosphate from growth factor enhanced matrix 21 (CaPfromGEM21, control), CaPfromGEM21 preloaded with 10 ng bone morphogenetic protein-2 (BMP-2), CaPfromGEM21 preloaded with 100 ng BMP-2, CaPfromGEM21 preloaded with 0.3 µg platelet-derived growth factor-BB (PDGF-BB), or CaPfromGEM21 preloaded with 3 µg PDGF-BB. The defects were examined 6 weeks after the surgery with X-ray, micro-CT, HE staining and quantitative assessments. X-ray showed defect repair in all the groups. The fracture line became obscure, and the defects were almost fully repaired by the regenerated bone tissues in PDGF-BB group. Micro-CT demonstarted new bone formation in the defects. The new bone volume was significantly greater in PDGF-BB groups than in BMP-2 groups (PBB group than in the control group (PBB has good biocompatibility and can better promote bone regeneration for repairing bone defects.

  17. Motorcycle Repair.

    Science.gov (United States)

    Hein, Jim; Bundy, Mike

    This motorcycle repair curriculum guide contains the following ten areas of study: brake systems, clutches, constant mesh transmissions, final drives, suspension, mechanical starting mechanisms, electrical systems, fuel systems, lubrication systems, and overhead camshafts. Each area consists of one or more units of instruction. Each instructional…

  18. Omphalocele repair

    Science.gov (United States)

    ... your child to visit a very ill sibling Surgical wound care - open Images Omphalocele repair - series References Chung DH. Pediatric surgery. In: Townsend CM Jr, Beauchamp RD, Evers BM, Mattox KL, ... Modern Surgical Practice . 20th ed. Philadelphia, PA: Elsevier; 2017:chap ...

  19. Turbine repair process, repaired coating, and repaired turbine component

    Science.gov (United States)

    Das, Rupak; Delvaux, John McConnell; Garcia-Crespo, Andres Jose

    2015-11-03

    A turbine repair process, a repaired coating, and a repaired turbine component are disclosed. The turbine repair process includes providing a turbine component having a higher-pressure region and a lower-pressure region, introducing particles into the higher-pressure region, and at least partially repairing an opening between the higher-pressure region and the lower-pressure region with at least one of the particles to form a repaired turbine component. The repaired coating includes a silicon material, a ceramic matrix composite material, and a repaired region having the silicon material deposited on and surrounded by the ceramic matrix composite material. The repaired turbine component a ceramic matrix composite layer and a repaired region having silicon material deposited on and surrounded by the ceramic matrix composite material.

  20. A microhomology-mediated break-induced replication model for the origin of human copy number variation.

    Directory of Open Access Journals (Sweden)

    P J Hastings

    2009-01-01

    Full Text Available Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV. A recent report of nonrecurrent duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2-5 base pairs (bp. Third, endpoints occur near pre-existing low copy repeats (LCRs. Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR. Under these circumstances, single-strand 3' tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication.

  1. Model Breaking Points Conceptualized

    Science.gov (United States)

    Vig, Rozy; Murray, Eileen; Star, Jon R.

    2014-01-01

    Current curriculum initiatives (e.g., National Governors Association Center for Best Practices and Council of Chief State School Officers 2010) advocate that models be used in the mathematics classroom. However, despite their apparent promise, there comes a point when models break, a point in the mathematical problem space where the model cannot,…

  2. Local Morphological Response of the Distal Femoral Articular–Epiphyseal Cartilage Complex of Young Foals to Surgical Stab Incision and Potential Relevance to Cartilage Injury and Repair in Children

    Science.gov (United States)

    Hendrickson, Eli H.S.; Ekman, Stina; Carlson, Cathy S.; Dolvik, Nils I.

    2013-01-01

    Objective: Describe the local morphological response of the articular–epiphyseal cartilage complex to surgical stab incision in the distal femur of foals, with emphasis on the relationship between growth cartilage injury, enchondral ossification, and repair. Design: Nine foals were induced into general anesthesia at the age of 13 to 15 days. Four full-thickness stab incision defects were created in the cartilage on the lateral aspect of the lateral trochlear ridge of the left distal femur. Follow-up examination was carried out from 1 to 49 days postoperatively, including examination of intact bones, sawed slabs, and histological sections. Results: Incision defects filled with cells displaying fibroblast-, chondrocyte-, and osteoblast-like characteristics, potentially validating the rationale behind the drilling of stable juvenile osteochondritis dissecans lesions in children. Incisions induced necrosis within the cartilage on the margins at all depths of the defects. Sharp dissection may therefore be contraindicated in cartilage repair in young individuals. Incisions caused a focal delay in enchondral ossification in 2 foals, apparently related to the orientation of the incision defect relative to the direction of ossification. Defects became progressively surrounded by subchondral bone, in which granulation tissue containing clasts and foci of osteoblast-like cells was observed. Continued enchondral ossification was therefore likely to result in healing of uncomplicated defects to morphologically normal bone. Conclusions: Epiphyseal growth cartilage injury had the potential to exert a negative effect on enchondral ossification. Enchondral ossification exerted a beneficial effect on repair. This relationship warrants consideration in future studies of cartilage injury and repair within the articular–epiphyseal cartilage complex of all species. PMID:26069670

  3. Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Martini

    2011-09-01

    Full Text Available Meiotic DNA double-strand breaks (DSBs initiate crossover (CO recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs. Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs. First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

  4. At the intersection of non-coding transcription, DNA repair, chromatin structure, and cellular senescence

    Directory of Open Access Journals (Sweden)

    Ryosuke eOhsawa

    2013-07-01

    Full Text Available It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence.

  5. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair.

    Science.gov (United States)

    Zahn, Karl E; Averill, April M; Aller, Pierre; Wood, Richard D; Doublié, Sylvie

    2015-04-01

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

  6. Bringing mask repair to the next level

    Science.gov (United States)

    Edinger, K.; Wolff, K.; Steigerwald, H.; Auth, N.; Spies, P.; Oster, J.; Schneider, H.; Budach, M.; Hofmann, T.; Waiblinger, M.

    2014-10-01

    Mask repair is an essential step in the mask manufacturing process as the extension of 193nm technology and the insertion of EUV are drivers for mask complexity and cost. The ability to repair all types of defects on all mask blank materials is crucial for the economic success of a mask shop operation. In the future mask repair is facing several challenges. The mask minimum features sizes are shrinking and require a higher resolution repair tool. At the same time mask blanks with different new mask materials are introduced to optimize optical performance and long term durability. For EUV masks new classes of defects like multilayer and phase defects are entering the stage. In order to achieve a high yield, mask repair has to cover etch and deposition capabilities and must not damage the mask. These challenges require sophisticated technologies to bring mask repair to the next level. For high end masks ion-beam based and e-based repair technologies are the obvious choice when it comes to the repair of small features. Both technologies have their pro and cons. The scope of this paper is to review and compare the performance of ion-beam based mask repair to e-beam based mask repair. We will analyze the limits of both technologies theoretically and experimentally and show mask repair related performance data. Based on this data, we will give an outlook to future mask repair tools.

  7. Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay.

    Science.gov (United States)

    Nickson, Catherine M; Parsons, Jason L

    2014-01-01

    Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20-50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A-DDB1-STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair

  8. Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

    Directory of Open Access Journals (Sweden)

    Jason Luke Parsons

    2014-07-01

    Full Text Available Base excision repair (BER is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionising radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation in the regulation of cellular levels of BER proteins, and that subtle changes (~20-50 % in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumour suppressor protein ARF and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A-DDB1-STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA

  9. [Neural repair].

    Science.gov (United States)

    Kitada, Masaaki; Dezawa, Mari

    2008-05-01

    Recent progress of stem cell biology gives us the hope for neural repair. We have established methods to specifically induce functional Schwann cells and neurons from bone marrow stromal cells (MSCs). The effectiveness of these induced cells was evaluated by grafting them either into peripheral nerve injury, spinal cord injury, or Parkinson' s disease animal models. MSCs-derived Schwann cells supported axonal regeneration and re-constructed myelin to facilitate the functional recovery in peripheral and spinal cord injury. MSCs-derived dopaminergic neurons integrated into host striatum and contributed to behavioral repair. In this review, we introduce the differentiation potential of MSCs and finally discuss about their benefits and drawbacks of these induction systems for cell-based therapy in neuro-traumatic and neuro-degenerative diseases.

  10. Balancing Pathways in DNA Double Strand Break Repair

    NARCIS (Netherlands)

    I. Brandsma (Inger)

    2016-01-01

    markdownabstractAll information a cell needs to live and survive is stored in the genomic DNA. Maintenance of an intact and uncompromised genome is of vital importance for cell survival. Damaged DNA can block transcription and replication, processes essential for cell viability. Persistent DNA

  11. Assaying Break and Nick-Induced Homologous Recombination in Mammalian Cells Using the DR-GFP Reporter and Cas9 Nucleases

    NARCIS (Netherlands)

    Vriend, Lianne E. M.; Jasin, Maria; Krawczyk, Przemek M.

    2014-01-01

    Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the

  12. Interference in DNA replication can cause mitotic chromosomal breakage unassociated with double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Mari Fujita

    Full Text Available Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs. We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways. Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/KU70(-/- DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/LIG4(-/- Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.

  13. Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae

    Science.gov (United States)

    Haber, James E.

    2016-01-01

    Correct repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Whereas gene conversion (GC)-mediated repair is mostly error-free, repair by break-induced replication (BIR) is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC) mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans) compared to the case when both DSB ends come from the same break (Cis). However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the “origin” of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6. PMID:27074148

  14. Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Suvi Jain

    2016-04-01

    Full Text Available Correct repair of DNA double-strand breaks (DSBs is critical for maintaining genome stability. Whereas gene conversion (GC-mediated repair is mostly error-free, repair by break-induced replication (BIR is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans compared to the case when both DSB ends come from the same break (Cis. However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the "origin" of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.

  15. DNA repair: keeping it together

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2004-01-01

    A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.......A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest....

  16. Breaking News as Radicalisation

    DEFF Research Database (Denmark)

    Hartley, Jannie Møller

    The aim of the paper is to make explicit how the different categories are applied in the online newsroom and thus how new categories can be seen as positioning strategies in the form of radicalisations of already existing categories. Thus field theory provides us with tools to analyse how online...... journalists are using the categorisations to create hierarchies within the journalistic field in order to position themselves as specialists in what Tuchman has called developing news, aiming and striving for what today is know as breaking news and the “exclusive scoop,” as the trademark of online journalism...... in a media environment where immediacy rules (Domingo 2008a). Following this research the primary focus of this paper is the category breaking news and Tuchmans developing news, but as they are all connected the analysis will also draw upon the other categories in Tuchmans typology. The theoretical framework...

  17. Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation.

    Science.gov (United States)

    Asaithamby, Aroumougame; Chen, David J

    2011-06-03

    Low-linear energy transfer (LET) radiation (i.e., γ- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles is slowly repaired or is irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure. 2010 Elsevier B.V. All rights reserved.

  18. Routinizing Breaking News

    DEFF Research Database (Denmark)

    Hartley, Jannie Møller

    2011-01-01

    This chapter revisits seminal theoretical categorizations of news proposed three decades earlier by US sociologist Gaye Tuchman. By exploring the definition of ”breaking news” in the contemporary online newsrooms of three Danish news organisations, the author offers us a long overdue re......-theorization of journalistic practice in the online context and helpfully explores well-evidenced limitations to online news production, such as the relationship between original reporting and the use of ”shovelware.”...

  19. Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Moore, Destaye M; Karlin, Justin; González-Barrera, Sergio

    2009-01-01

    . Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB......In the yeast Saccharomyces cerevisiae, the Rad1-Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1-Rad10 endonuclease cleaves 3' branches of DNA and aberrant 3' DNA ends that are refractory to other 3' processing enzymes...

  20. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wessendorf, Petra [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany); Vijg, Jan [Albert Einstein College of Medicine, Michael F. Price Center, 1301 Morris Park Avenue, Bronx, NY 10461 (United States); Nussenzweig, André [Laboratory of Genome Integrity, National Cancer Institute, National Institute of Health, 37 Convent Drive, Room 1106, Bethesda, MD 20892 (United States); Digweed, Martin, E-mail: martin.digweed@charite.de [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany)

    2014-11-15

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.

  1. Hybrid Repair of Complex Thoracic Aortic Arch Pathology: Long-Term Outcomes of Extra-anatomic Bypass Grafting of the Supra-aortic Trunk

    Energy Technology Data Exchange (ETDEWEB)

    Lotfi, S., E-mail: shamim.lotfi@kcl.ac.uk; Clough, R. E.; Ali, T. [Guy' s and St. Thomas' NHS Trust, Vascular Surgery (United Kingdom); Salter, R. [Guy' s and St. Thomas' NHS Trust, Interventional Radiology (United Kingdom); Young, C. P. [Guy' s and St. Thomas' NHS Trust, Cardiac Surgery (United Kingdom); Bell, R.; Modarai, B.; Taylor, P., E-mail: peter.taylor@gstt.nhs.uk [Guy' s and St. Thomas' NHS Trust, Vascular Surgery (United Kingdom)

    2013-02-15

    Hybrid repair constitutes supra-aortic debranching before thoracic endovascular aortic repair (TEVAR). It offers improved short-term outcome compared with open surgery; however, longer-term studies are required to assess patient outcomes and patency of the extra-anatomic bypass grafts. A prospectively maintained database of 380 elective and urgent patients who had undergone TEVAR (1997-2011) was analyzed retrospectively. Fifty-one patients (34 males; 17 females) underwent hybrid repair. Median age was 71 (range, 18-90) years with mean follow-up of 15 (range, 0-61) months. Perioperative complications included death: 10 % (5/51), stroke: 12 % (6/51), paraplegia: 6 % (3/51), endoleak: 16 % (8/51), rupture: 4 % (2/51), upper-limb ischemia: 2 % (1/51), bypass graft occlusion: 4 % (2/51), and cardiopulmonary complications in 14 % (7/51). Three patients (6 %) required emergency intervention for retrograde dissection: (2 aortic root repairs; 2 innominate stents). Early reintervention was performed for type 1 endoleak in two patients (2 proximal cuff extensions). One patient underwent innominate stenting and revision of their bypass for symptomatic restenosis. At 48 months, survival was 73 %. Endoleak was detected in three (6 %) patients (type 1 = 2; type 2 = 1) requiring debranching with proximal stent graft (n = 2) and proximal extension cuff (n = 1). One patient had a fatal rupture of a mycotic aneurysm and two arch aneurysms expanded. No bypass graft occluded after the perioperative period. Hybrid operations to treat aortic arch disease can be performed with results comparable to open surgery. The longer-term outcomes demonstrate low rates of reintervention and high rates of graft patency.

  2. Double-strand breaks and the concept of short- and long-term epigenetic memory.

    Science.gov (United States)

    Orlowski, Christian; Mah, Li-Jeen; Vasireddy, Raja S; El-Osta, Assam; Karagiannis, Tom C

    2011-04-01

    Double-strand breaks represent an extremely cytolethal form of DNA damage and thus pose a serious threat to the preservation of genetic and epigenetic information. Though it is well-known that double-strand breaks such as those generated by ionising radiation are among the principal causative factors behind mutations, chromosomal aberrations, genetic instability and carcinogenesis, significantly less is known about the epigenetic consequences of double-strand break formation and repair for carcinogenesis. Double-strand break repair is a highly coordinated process that requires the unravelling of the compacted chromatin structure to facilitate repair machinery access and then restoration of the original undamaged chromatin state. Recent experimental findings have pointed to a potential mechanism for double-strand break-induced epigenetic silencing. This review will discuss some of the key epigenetic regulatory processes involved in double-strand break (DSB) repair and how incomplete or incorrect restoration of chromatin structure can leave a DSB-induced epigenetic memory of damage with potentially pathological repercussions.

  3. Complexity

    Indian Academy of Sciences (India)

    Rahul Pandit

    2008-10-31

    Oct 31, 2008 ... ”The more complex a thing is, the more you can talk about it.” - attributed to Giorgio Parisi. ▻ ”C'est magnifique, mais ce n'est pas de la science.” (It is magnificent, but not all of it is science.) - attributed ... Earliest examples: theoretical computer science, algorithmic complexity, etc. ▻ Rapid progress after the ...

  4. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    Science.gov (United States)

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  5. Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair.

    Science.gov (United States)

    Nassif, N; Penney, J; Pal, S; Engels, W R; Gloor, G B

    1994-03-01

    P-element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8,000 bp of nonhomologous sequence could be copied from an ectopic template at essentially the same rate as a single-base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila melanogaster, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as a template. This synthesis-dependent strand annealing (SDSA) model as a possible general mechanism in complex organisms is discussed.

  6. DNA repair, damage signaling and carcinogenesis.

    Science.gov (United States)

    Lavelle, Christophe; Salles, Bernard; Wiesmüller, Lisa

    2008-04-02

    The First joint meeting of the German DGDR (German Society for Research on DNA Repair) and the French SFTG (French Society of Genotoxicology) on DNA Repair was held in Toulouse, France, from September 15 to 19, 2007. It was organized by Lisa Wiesmüller and Bernard Salles together with the scientific committee consisting of Gilbert de Murcia, Jean-Marc Egly, Frank Grosse, Karl-Peter Hopfner, Georges Iliakis, Bernd Kaina, Markus Löbrich, Bernard Lopez, Daniel Marzin and Alain Sarasin. This report summarizes information presented by the speakers (invited lectures and oral communications) during the seven plenary sessions, which include (1) excision repair, (2) DNA repair and carcinogenesis, (3) double-strand break repair, (4) replication in repair and lesion bypass, (5) cellular responses to genotoxic stress, (6) DNA repair machinery within the chromatin context and (7) genotoxicology and testing. A total of 23 plenary lectures, 32 oral communications and 66 posters were presented in this rather intense 4 days meeting, which stimulated extensive discussions and highly interdisciplinary scientific exchanges among the approximately 250 participants.

  7. Break the Pattern!

    DEFF Research Database (Denmark)

    Hasse, Cathrine; Trentemøller, Stine

    Break the Pattern! A critical enquiry into three scientific workplace cultures: Hercules, Caretakers and Worker Bees is the third publication of the international three year long project "Understanding Puzzles in the Gendered European Map" (UPGEM). By contrasting empirical findings from academic ...... (physics in culture) and discuss how physics as and in culture influence the perception of science, of work and family life, of the interplay between religion and science as well as how physics as culture can either hinder or promote the career of female scientists....

  8. Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair

    Directory of Open Access Journals (Sweden)

    Tony Gutschner

    2016-02-01

    Full Text Available CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ, or homology-directed repair (HDR. While HDR is absent in G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell-cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1, resulting in a cell-cycle-tailored expression with low levels in G1 but high expression in S/G2/M. Importantly, Cas9-hGem(1/110 increased the rate of HDR by up to 87% compared to wild-type Cas9. Future developments may enable high-resolution expression of genome engineering proteins, which might increase HDR rates further, and may contribute to a better understanding of DNA repair pathways due to spatiotemporal control of DNA damage induction.

  9. Brain aneurysm repair

    Science.gov (United States)

    ... aneurysm repair; Dissecting aneurysm repair; Endovascular aneurysm repair - brain; Subarachnoid hemorrhage - aneurysm ... Your scalp, skull, and the coverings of the brain are opened. A metal clip is placed at ...

  10. Eye muscle repair - discharge

    Science.gov (United States)

    ... Lazy eye repair - discharge; Strabismus repair - discharge; Extraocular muscle surgery - discharge ... You or your child had eye muscle repair surgery to correct eye muscle ... term for crossed eyes is strabismus. Children most often ...

  11. Inguinal hernia repair

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007406.htm Inguinal hernia repair To use the sharing features on this ... Inguinal hernia repair is surgery to repair a hernia in your groin. A hernia is tissue that bulges out of ...

  12. Femoral hernia repair

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/007399.htm Femoral hernia repair To use the sharing features on this page, please enable JavaScript. Femoral hernia repair is surgery to repair a hernia near ...

  13. A retrospective evaluation of the aesthetics of the nasolabial complex after unilateral cleft lip repair using the Tennison-Randall technique: a study of 44 cases treated in a single cleft center.

    Science.gov (United States)

    Iliopoulos, Christos; Mitsimponas, Konstantinos; Lazaridou, Dimitra; Neukam, Friedrich Wilhelm; Stelzle, Florian

    2014-12-01

    Among numerous techniques that have been described for lip repair, the Tennison-Randall method has gained popularity over time and is preferred by many surgeons due to the predictability of the outcome. This study aims to evaluate the esthetic outcome reached in the nasolabial region following primary lip repair with the use of this method. Forty-four patients with unilateral cleft lip (with or without alveolar cleft) were assessed retrospectively through a photographic evaluation by two clinicians with regard to the aesthetics of the lip and nose separately as anatomical subunits as well as of the nasolabial region as an anatomical complex. The collected data were statistically analyzed with regard to the cleft subtype and the performance of corrective surgeries for the lip and/or the nose. The method was associated with good results, especially when it comes to the appearance of the nose as an anatomical subunit, as well as of the nasolabial region as a complex, regarding cleft lip patients without an alveolar cleft. The Tennison-Randall technique proved to be a very satisfying method in terms of the esthetic long-term outcome in our patient collective. Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  14. Peripherally Inserted Central Catheters in Pediatric Patients: To Repair or Not Repair

    Energy Technology Data Exchange (ETDEWEB)

    Gnannt, Ralph, E-mail: ralph.gnannt@usz.ch; Patel, Premal; Temple, Michael; Al Brashdi, Yahya; Amaral, Joao; Parra, Dimitri; Rea, Vanessa [University of Toronto, Image Guided Therapy, Diagnostic Imaging, The Hospital for Sick Children (Canada); Stephens, Derek [University of Toronto, Child Health Evaluative Sciences (Canada); Connolly, Bairbre [University of Toronto, Image Guided Therapy, Diagnostic Imaging, The Hospital for Sick Children (Canada)

    2017-06-15

    IntroductionPreservation of venous access in children is a major concern in pediatric interventional radiology. If a peripherally inserted central catheter (PICC) breaks, there are two options: repair the line with a repair kit or exchange the line over a wire in the interventional suite. The purpose of this study is to assess the outcome of PICC repairs in children and to compare these with the outcomes of PICC exchange.Materials and MethodsThis is a single-center, retrospective study of central line-associated bloodstream infection (CLABSI) following management of externally broken PICCs (2010–2014). The occurrence of CLABSI within 30 days after repair (Group A) or exchange (Group B) of a line was analyzed, as well as PICCs exchanged following an initial and failed repair.ResultsA total of 235 PICC breaks were included in the study, of which 161 were repaired, and 116 of whom were successful (68%, Group A). No repair was performed in 74 PICCs—55/74 of these were exchanged over a wire (74%, Group B), and 19/74 lines were removed. The 30 days post-repair CLABSI rate (Group A) was 2.0 infections per 1000 catheter days, and the calculated risk was 4.3%. In comparison the 30 days post-exchange CLABSI rate (Group B) was 4.0 per 1000 catheter days and the calculated risk 10.9%. This difference was significant when adjusted for antibiotic use (OR 3.87; 95% CI 1.07–14.0, p = 0.039).ConclusionThe results of this study support repairing a broken PICC instead of removing or replacing the line.

  15. Defects on endoanal ultrasound and anal incontinence after primary repair of fourth-degree anal sphincter rupture: a study of the anal sphincter complex and puborectal muscle

    DEFF Research Database (Denmark)

    Sakse, A; Secher, N J; Ottesen, M

    2009-01-01

    OBJECTIVES: To perform three-dimensional endoanal ultrasound (EAUS) after primary repair of fourth-degree anal sphincter rupture (ASR) and correlate the sonographic defects with anal incontinence (AI); to measure the axial and sagittal thickness and angle of the puborectal muscle (PRM) as well...... as the length of the anal canal, and then correlate these measures with AI; and to assess the interobserver measurement agreement between an inexperienced and an experienced sonologist. METHODS: EAUS was offered to 84 consecutive women, who were asked to answer a validated questionnaire after fourth-degree ASR...

  16. Bootstrap Dynamical Symmetry Breaking

    Directory of Open Access Journals (Sweden)

    Wei-Shu Hou

    2013-01-01

    Full Text Available Despite the emergence of a 125 GeV Higgs-like particle at the LHC, we explore the possibility of dynamical electroweak symmetry breaking by strong Yukawa coupling of very heavy new chiral quarks Q . Taking the 125 GeV object to be a dilaton with suppressed couplings, we note that the Goldstone bosons G exist as longitudinal modes V L of the weak bosons and would couple to Q with Yukawa coupling λ Q . With m Q ≳ 700  GeV from LHC, the strong λ Q ≳ 4 could lead to deeply bound Q Q ¯ states. We postulate that the leading “collapsed state,” the color-singlet (heavy isotriplet, pseudoscalar Q Q ¯ meson π 1 , is G itself, and a gap equation without Higgs is constructed. Dynamical symmetry breaking is affected via strong λ Q , generating m Q while self-consistently justifying treating G as massless in the loop, hence, “bootstrap,” Solving such a gap equation, we find that m Q should be several TeV, or λ Q ≳ 4 π , and would become much heavier if there is a light Higgs boson. For such heavy chiral quarks, we find analogy with the π − N system, by which we conjecture the possible annihilation phenomena of Q Q ¯ → n V L with high multiplicity, the search of which might be aided by Yukawa-bound Q Q ¯ resonances.

  17. Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in Schizosaccharomyces pombe.

    Science.gov (United States)

    Li, Yanhui; Wang, Jinyu; Zhou, Gang; Lajeunesse, Michael; Le, Nga; Stawicki, Brittany N; Corcino, Yalitza Lopez; Berkner, Kathleen L; Runge, Kurt W

    2017-05-01

    While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in nonhomologous end-joining (NHEJ) is unclear as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mammals have different requirements for repairing cut DNA ends. Most double-strand breaks (DSBs) require nucleolytic processing prior to DNA ligation. Therefore, we studied repair using the Hermes transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single Hermes insertions using a novel S. pombe transient transfection system, and used Hermes excision to show a requirement for MRN in the NHEJ of nonligatable ends. NHEJ repair was indicated by the >1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had pombe NHEJ was reduced >1000-fold in cells lacking each MRN subunit, and loss of MRN-associated Ctp1 caused a 30-fold reduction. An Mre11 dimer is thought to hold DNA ends together for repair, and Mre11 dimerization domain mutations reduced repair 300-fold. In contrast, a mre11 mutant defective in endonucleolytic activity, the same mutant lacking Ctp1, or the triple mutant also lacking the putative hairpin nuclease Pso2 showed wild-type levels of repair. Thus, MRN may act to recruit the hairpin opening activity that allows subsequent repair. Copyright © 2017 by the Genetics Society of America.

  18. Modulated Tool-Path (MTP) Chip Breaking System

    Energy Technology Data Exchange (ETDEWEB)

    Graham, K. B.

    2010-04-01

    The Modulated Tool-Path (MTP) Chip Breaking System produces user-selectable chip lengths and workpiece finishes and is compatible with any material, workpiece shape, and depth of cut. The MTP chip breaking system consistently creates the desired size of chips regardless of workpiece size, shape, or material, and the machine operator does not need to make any adjustments during the machining operation. The system's programmer configures the part program that commands the machine tool to move in a specific fashion to deliver the desired part size, shape, chip length, and workpiece surface finish. The MTP chip breaking system helps manufacturers avoid the detrimental effects of continuous chips, including expensive repair costs, delivery delays, and hazards to personnel.

  19. Mismatch repair.

    Science.gov (United States)

    Fishel, Richard

    2015-10-30

    Highly conserved MutS homologs (MSH) and MutL homologs (MLH/PMS) are the fundamental components of mismatch repair (MMR). After decades of debate, it appears clear that the MSH proteins initiate MMR by recognizing a mismatch and forming multiple extremely stable ATP-bound sliding clamps that diffuse without hydrolysis along the adjacent DNA. The function(s) of MLH/PMS proteins is less clear, although they too bind ATP and are targeted to MMR by MSH sliding clamps. Structural analysis combined with recent real-time single molecule and cellular imaging technologies are providing new and detailed insight into the thermal-driven motions that animate the complete MMR mechanism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Mismatch Repair*

    Science.gov (United States)

    Fishel, Richard

    2015-01-01

    Highly conserved MutS homologs (MSH) and MutL homologs (MLH/PMS) are the fundamental components of mismatch repair (MMR). After decades of debate, it appears clear that the MSH proteins initiate MMR by recognizing a mismatch and forming multiple extremely stable ATP-bound sliding clamps that diffuse without hydrolysis along the adjacent DNA. The function(s) of MLH/PMS proteins is less clear, although they too bind ATP and are targeted to MMR by MSH sliding clamps. Structural analysis combined with recent real-time single molecule and cellular imaging technologies are providing new and detailed insight into the thermal-driven motions that animate the complete MMR mechanism. PMID:26354434

  1. Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

    Science.gov (United States)

    Ma, Xiao-Juan; Shang, Li; Zhang, Wei-Min; Wang, Ming-Rong; Zhan, Qi-Min

    2016-04-10

    Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. D-ribose inhibits DNA repair synthesis in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zunica, G.; Marini, M.; Brunelli, M.A.; Chiricolo, M.; Franceschi, C.

    1986-07-31

    D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.

  3. Cell birth, cell death, cell diversity and DNA breaks: how do they all fit together?

    Science.gov (United States)

    Gilmore, E. C.; Nowakowski, R. S.; Caviness, V. S. Jr; Herrup, K.

    2000-01-01

    Substantial death of migrating and differentiating neurons occurs within the developing CNS of mice that are deficient in genes required for repair of double-stranded DNA breaks. These findings suggest that large-scale, yet previously unrecognized, double-stranded DNA breaks occur normally in early postmitotic and differentiating neurons. Moreover, they imply that cell death occurs if the breaks are not repaired. The cause and natural function of such breaks remains a mystery; however, their occurrence has significant implications. They might be detected by histological methods that are sensitive to DNA fragmentation and mistakenly interpreted to indicate cell death when no relationship exists. In a broader context, there is now renewed speculation that DNA recombination might be occurring during neuronal development, similar to DNA recombination in developing lymphocytes. If this is true, the target gene(s) of recombination and their significance remain to be determined.

  4. Energy and Technology Review: Unlocking the mysteries of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  5. Prophylactic treatment of retinal breaks

    DEFF Research Database (Denmark)

    Blindbæk, Søren Leer; Grauslund, Jakob

    2015-01-01

    Prophylactic treatment of retinal breaks has been examined in several studies and reviews, but so far, no studies have successfully applied a systematic approach. In the present systematic review, we examined the need of follow-up after posterior vitreous detachment (PVD) - diagnosed by slit...... breaks. Additional retinal breaks were only revealed at follow-up in patients where a full retinal examination was compromised at presentation by, for example, vitreous haemorrhage. Asymptomatic and symptomatic retinal breaks progressed to rhegmatogenous retinal detachment (RRD) in 0-13.8% and 35...

  6. Electroweak breaking in supersymmetric models

    CERN Document Server

    Ibáñez, L E

    1992-01-01

    We discuss the mechanism for electroweak symmetry breaking in supersymmetric versions of the standard model. After briefly reviewing the possible sources of supersymmetry breaking, we show how the required pattern of symmetry breaking can automatically result from the structure of quantum corrections in the theory. We demonstrate that this radiative breaking mechanism works well for a heavy top quark and can be combined in unified versions of the theory with excellent predictions for the running couplings of the model. (To be published in ``Perspectives in Higgs Physics'', G. Kane editor.)

  7. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    Energy Technology Data Exchange (ETDEWEB)

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  8. The predicted truncation from a cancer-associated variant of the MSH2 initiation codon alters activity of the MSH2-MSH6 mismatch repair complex.

    Science.gov (United States)

    Cyr, Jennifer L; Brown, Graham D; Stroop, Jennifer; Heinen, Christopher D

    2012-08-01

    Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes. MMR recognizes and repairs DNA mismatches and small insertion/deletion loops. Carriers of MMR gene variants have a high risk of developing colorectal, endometrial, ovarian, and other extracolonic carcinomas. We report on an ovarian cancer patient who carries a germline MSH2 c.1A>C variant which alters the translation initiation codon. Mutations affecting the MSH2 start codon have been described previously for LS-related malignancies. However, the patients often lack a clear family history indicative of LS and their tumors often fail to display microsatellite instability, a hallmark feature of LS. Therefore, the pathogenicity of start codon variants remains undefined. Loss of the MSH2 start codon has been predicted to result in a truncated protein translated from a downstream in-frame AUG that would lack the first 25 amino acids. We therefore purified recombinant MSH2(NΔ25)-MSH6 and MSH2(NΔ25)-MSH3 to examine their DNA lesion recognition and adenosine nucleotide processing functions in vitro. We found that the MSH2(NΔ25) mutant confers distinct biochemical defects on MSH2-MSH6, but does not have a significant effect on MSH2-MSH3. We confirmed that expression of the MSH2 c.1A>C cDNA results in the production of multiple protein products in human cells that may include the truncated and full-length forms of MSH2. An in vivo MMR assay revealed a slight reduction in MMR efficiency in these cells. These data suggest that mutation of the MSH2 initiation codon, while not a strong, high-risk disease allele, may have a moderate impact on disease phenotype. Copyright © 2011 Wiley Periodicals, Inc.

  9. Breaking soliton equations and negative-order breaking soliton ...

    Indian Academy of Sciences (India)

    We develop breaking soliton equations and negative-order breaking soliton equations of typical and higher orders. The recursion operator of the KdV equation is used to derive these models.We establish the distinctdispersion relation for each equation. We use the simplified Hirota's method to obtain multiple soliton ...

  10. EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair.

    Directory of Open Access Journals (Sweden)

    Yuehan Wu

    2015-12-01

    Full Text Available Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR or non-conservatively repaired using micro-homology mediated end joining (MMEJ. HR repair of stressed forks is initiated by 5' end resection near the fork junction, which permits 3' single strand invasion of a homologous template for fork restart. This 5' end resection also prevents classical non-homologous end-joining (cNHEJ, a competing pathway for DNA double-strand break (DSB repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1 protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5' DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5' overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.

  11. DNA Repair Systems

    Indian Academy of Sciences (India)

    nal factors such as UV radiation, high energy radiation such as X-. Keywords. DNA repair, DNA damage, base excision repair, nucleotide exci- sion repair, methlyl-directed mis- match repair, Nobel Prize. rays and gamma rays, mutagenic chemicals and viruses. Different types of DNA ... be especially important in plants.

  12. DNA Repair Dysfunction and Neurodegeneration: Lessons From Rare Pediatric Disorders.

    Science.gov (United States)

    Shabbir, Syed H

    2016-03-01

    Nucleotide excision repair disorders display a wide range of clinical syndromes and presentations, all associated at the molecular level by dysfunction of genes participating in the nucleotide excision repair pathway. Genotype-phenotype relationships are remarkably complex and not well understood. This article outlines neurodegenerative symptoms seen in nucleotide excision repair disorders and explores the role that nucleotide excision repair dysfunction can play in the pathogenesis of chronic neurodegenerative diseases. © The Author(s) 2015.

  13. Non-minimal scalar multiplets, supersymmetry breaking and dualities

    Energy Technology Data Exchange (ETDEWEB)

    Farakos, Fotis; Hulík, Ondřej; Kočí, Pavel; Unge, Rikard von [Institute for Theoretical Physics, Masaryk University,611 37 Brno (Czech Republic)

    2015-09-25

    We study supersymmetry breaking in theories with non-minimal multiplets (such as the complex linear or CNM multiplets), by using superspace higher derivative terms which give rise to new supersymmetry breaking vacuum solutions on top of the standard supersymmetric vacuum. We illustrate the decoupling of the additional massive sectors inside the complex linear and the CNM multiplets and show that only the Goldstino sector is left in the low energy limit. We also discuss the duality between non-minimal scalar multiplets and chiral multiplets in the presence of superspace higher derivatives. From the superspace Noether procedure we calculate the supercurrents, and we show that in the supersymmetry breaking vacuum the chiral superfield X which enters the Ferrara-Zumino supercurrent conservation equation does indeed flow in the IR to the chiral constrained Goldstino superfield. We also provide a description of the Goldstino sector in terms of the Samuel-Wess superfield for the supersymmetry breaking mechanism at hand.

  14. Relocalization of DNA lesions to the nuclear pore complex.

    Science.gov (United States)

    Freudenreich, Catherine H; Su, Xiaofeng A

    2016-12-01

    Early screens in yeast for mutations exhibiting sensitivity to DNA damage identified nuclear pore components, but their role in DNA repair was not well understood. Over the last decade, studies have revealed that several types of persistent DNA lesions relocate to either the nuclear pore complex (NPC) or nuclear envelope (NE). Of these two sites, the nuclear pore appears to be crucial for DNA repair of persistent double-strand breaks, eroded telomeres and sites of fork collapse at expanded CAG repeats. Using a combination of cell biological imaging techniques and yeast genetic assays for DNA repair, researchers have begun to understand both the how and why of lesion relocation to the NPC. Here we review the types of lesions that relocate to the NPC, mediators of relocation and the functional consequences of relocation understood to date. The emerging theme is that relocation to the NPC regulates recombination to influence repair pathway choice and provide a rescue mechanism for lesions or DNA structures that are resistant to repair. © FEMS 2016.

  15. Core break-off mechanism

    Science.gov (United States)

    Myrick, Thomas M. (Inventor)

    2003-01-01

    A mechanism for breaking off and retaining a core sample of a drill drilled into a ground substrate has an outer drill tube and an inner core break-off tube sleeved inside the drill tube. The break-off tube breaks off and retains the core sample by a varying geometric relationship of inner and outer diameters with the drill tube. The inside diameter (ID) of the drill tube is offset by a given amount with respect to its outer diameter (OD). Similarly, the outside diameter (OD) of the break-off tube is offset by the same amount with respect to its inner diameter (ID). When the break-off tube and drill tube are in one rotational alignment, the two offsets cancel each other such that the drill can operate the two tubes together in alignment with the drill axis. When the tubes are rotated 180 degrees to another positional alignment, the two offsets add together causing the core sample in the break-off tube to be displaced from the drill axis and applying shear forces to break off the core sample.

  16. Spontaneous Symmetry Breaking in Nonrelativistic Systems

    Science.gov (United States)

    Watanabe, Haruki

    The subject of condensed matter physics is very rich --- there are an infinite number of parameters producing a diversity of exciting phenomena. As a theorist, my goal is to distill general principles out of this complexity --- to construct theories that can coherently explain many known examples altogether. This thesis is composed of several attempts to develop such theories in topics related to spontaneously symmetry breaking. A remarkable feature of many-body interacting systems is that although they are described by equations respecting various symmetries, they may spontaneously organize into a state that explicitly breaks symmetries. Examples are numerous: various types of crystalline and magnetic orders, Bose-Einstein condensates of cold atoms, superfluids of liquid helium, chiral symmetry in QCD, neutron stars, and cosmic inflation. These systems with spontaneously broken continuous symmetries have gapless excitations, so called Nambu-Goldstone bosons (NGBs). Although the properties of NGBs are well understood in Lorentz-invariant systems, surprisingly, some basic properties of NGBs such as their number and dispersion in nonrelativistic systems have not been discussed from a general perspective. In the first part of this thesis, we solve this issue by developing and analyzing an effective Lagrangian that coherently captures the low-energy, long-distance physics of many different symmetry-breaking states all at once. Next, we examine whether these NGBs originating from spontaneous symmetry breaking remain to be well-defined excitations inside a metal, where low-energy electrons near Fermi surface can collide with them. Our result is a one equation criterion that specifies whether the interactions between electrons and NGBs can be ignored, or whether it completely changes their character. In the latter case, unusual phases of matter such as non-Fermi liquids may arise; in that case, NGBs are overdamped and cannot form particle-like excitations in spite of the

  17. Signalling of double strand breaks and deprotected telomeres in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Simon eAmiard

    2013-10-01

    Full Text Available Failure to repair DNA double strand breaks (DSB can lead to chromosomal rearrangements and eventually to cancer or cell death. Radiation and environmental pollutants induce DSB and this is of particular relevance to plants due to their sessile life style. DSB also occur naturally in cells during DNA replication and programmed induction of DSB initiates the meiotic recombination essential for gametogenesis in most eukaryotes. The linear nature of most eukaryotic chromosomes means that each chromosome has two "broken" ends. Chromosome ends, or telomeres, are protected by nucleoprotein caps which avoid their recognition as DSB by the cellular DNA repair machinery. Deprotected telomeres are recognized as DSB and become substrates for recombination leading to chromosome fusions, the "bridge-breakage-fusion" cycle, genome rearrangements and cell death. The importance of repair of DSB and the severity of the consequences of their misrepair have led to the presence of multiple, robust mechanisms for their detection and repair. After a brief overview of DSB repair pathways to set the context, we present here an update of current understanding of the detection and signalling of DSB in the plant, Arabidopsis thaliana.

  18. Role of Rad54, Rad54b and Snm1 in DNA damage repair

    NARCIS (Netherlands)

    J. Wesoly (Joanna)

    2003-01-01

    textabstractThe aim of this thesis is to investigate the function of a number of genes involved in mammalian DNA damage repair, in particular in repair of DNA double-strand breaks (DSBs). Among a large number of different damages that can be introduced to DNA, DSBs are especially toxic. If

  19. Catheter Ablation of a Complex Atrial Tachycardia after Surgical Repair of Tetralogy of Fallot Guided by Combined Noncontact and Contact Mapping

    Directory of Open Access Journals (Sweden)

    Eitaro Fujii, MD

    2010-01-01

    Full Text Available A 34-year-old man with a surgically repaired Tetralogy of Fallot complained of palpitation, fatigue, and presyncope. A 12-lead ECG showed atrial tachycardia with a cycle length of 250 ms and a P wave morphology positive in leads II, III and aVF, and negative in lead V1. Although the EnSite system (version 6.OJ made use of noncontact mapping to delineate the counterclockwise reentry around the crista tenninalis, it was difficult to rule out the incisional atrial reentry because the location of the surgical incision was far from the multi-electrode array. Since the bipolar contact mapping of the EnSite system revealed the location of the atriotomy incision, entrainment mapping during the tachycardia demonstrated the critical reentry circuit around the crista terminalis. Radiofrequency ablation targeting the critical isthmus from the lower position of the crista terminalis to the posterior dense scar which was continuous with the inferior vena cava, and to the atriotomy scar, eliminated the tachycardia.

  20. Making Weak Bonds (cooling) and Breaking Strong Bonds (heating ...

    Indian Academy of Sciences (India)

    Making Weak Bonds (cooling) and Breaking Strong Bonds (heating) with Supersonic Techniques · Acknowledgements · Outline · Slide 4 · A comparison of the two techniques · What is a hydrogen bond? Phenylacetylene and its complexes · Slide 8 · Phenylacetylene and PAH · phenylacetylene · Complexes in the interstellar ...

  1. The Deubiquitylating Enzyme USP4 Cooperates with CtIP in DNA Double-Strand Break End Resection

    Directory of Open Access Journals (Sweden)

    Hailong Liu

    2015-10-01

    Full Text Available DNA end resection is a highly regulated and critical step in DNA double-stranded break (DSB repair. In higher eukaryotes, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN complex. Here, we find that the deubiquitylating enzyme USP4 directly participates in DSB resection and homologous recombination (HR. USP4 confers resistance to DNA damage-inducing agents. Mechanistically, USP4 interacts with CtIP and MRN via a specific, conserved region and the catalytic domain of USP4, respectively, and regulates CtIP recruitment to sites of DNA damage. We also find that USP4 autodeubiquitylation is essential for its HR functions. Collectively, our findings identify USP4 as a key regulator of DNA DSB end resection.

  2. 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.

  3. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    Science.gov (United States)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy. Copyright © 2014. Published by Elsevier B.V.

  4. The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion

    NARCIS (Netherlands)

    Dessing, M.C.; Tammaro, A.; Pulskens, W.P.C.; Teske, G.J.; Butter, L.M.; Claessen, N.; Eijk, M. van; Poll, T. van der; Vogl, T.; Roth, J.; Florquin, S.; Leemans, J.C.

    2015-01-01

    Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury.

  5. A Study of BMP-2-Loaded Bipotential Electrolytic Complex around a Biphasic Calcium Phosphate-Derived (BCP Scaffold for Repair of Large Segmental Bone Defect.

    Directory of Open Access Journals (Sweden)

    Kallyanashis Paul

    Full Text Available A bipotential polyelectrolyte complex with biphasic calcium phosphate (BCP powder dispersion provides an excellent option for protein adsorption and cell attachment and can facilitate enhanced bone regeneration. Application of the bipotential polyelectrolyte complex embedded in a spongy scaffold for faster healing of large segmental bone defects (LSBD can be a promising endeavor in tissue engineering application. In the present study, a hollow scaffold suitable for segmental long bone replacement was fabricated by the sponge replica method applying the microwave sintering process. The fabricated scaffold was coated with calcium alginate at the shell surface, and genipin-crosslinked chitosan with biphasic calcium phosphate (BCP dispersion was loaded at the central hollow core. The chitosan core was subsequently loaded with BMP-2. The electrolytic complex was characterized using SEM, porosity measurement, FTIR spectroscopy and BMP-2 release for 30 days. In vitro studies such as MTT, live/dead, cell proliferation and cell differentiation were performed. The scaffold was implanted into a 12 mm critical size defect of a rabbit radius. The efficacy of this complex is evaluated through an in vivo study, one and two month post implantation. BV/TV ratio for BMP-2 loaded sample was (42±1.76 higher compared with hollow BCP scaffold (32±0.225.

  6. Battlefield Damage Assessment and Repair: Is Improvised Maintenance the Battlefield Solution to the Repair Parts Dilemma

    Science.gov (United States)

    1993-12-14

    decade, under Secretary of Defense Robert S. McNamara, operations research (specifically, cost-benefit 8 analysis) became a primary tool for military...be preferable: Equating A. and CA., using x to represent the break-even BDAR capability may be represented by the equation: Ao=40%= (10 houzs - I hour...commanders to overcome the problems of non-availability of repair parts through "[scrounging] and cannibalization." 38. McFarlin, Robert P., Memorandum

  7. Effect of Amalaki rasayana on DNA damage and repair in randomized aged human individuals.

    Science.gov (United States)

    Vishwanatha, Udupi; Guruprasad, Kanive P; Gopinath, Puthiya M; Acharya, Raviraj V; Prasanna, Bokkasa V; Nayak, Jayakrishna; Ganesh, Rajeshwari; Rao, Jayalaxmi; Shree, Rashmi; Anchan, Suchitra; Raghu, Kothanahalli S; Joshi, Manjunath B; Paladhi, Puspendu; Varier, Panniampilly M; Muraleedharan, Kollath; Muraleedharan, Thrikovil S; Satyamoorthy, Kapaettu

    2016-09-15

    Preparations from Phyllanthus emblica called Amalaki rasayana is used in the Indian traditional medicinal system of Ayurveda for healthy living in elderly. The biological effects and its mechanisms are not fully understood. Since the diminishing DNA repair is the hallmark of ageing, we tested the influence of Amalaki rasayana on recognized DNA repair activities in healthy aged individuals. Amalaki rasayana was prepared fresh and healthy aged randomized human volunteers were administrated with either rasayana or placebo for 45 days strictly as per the traditional text. The DNA repair was analyzed in peripheral blood mononuclear cells before and after rasayana administration and after 45 days post-rasayana treatment regimen. UVC-induced DNA strand break repair (DSBR) based on extent of DNA unwinding by fluorometric analysis, nucleotide excision repair (NER) by flow cytometry and constitutive base excision repair (BER) by gap filling method were analyzed. Amalaki rasayana administration stably maintained/enhanced the DSBR in aged individuals. There were no adverse side effects. Further, subjects with different body mass index showed differential DNA strand break repair capacity. No change in unscheduled DNA synthesis during NER and BER was observed between the groups. Intake of Amalaki rasayana by aged individuals showed stable maintenance of DNA strand break repair without toxic effects. However, there was no change in nucleotide and base excision repair activities. Results warrant further studies on the effects of Amalaki rasayana on DSBR activities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. ATM-Dependent Phosphorylation of All Three Members of the MRN Complex: From Sensor to Adaptor.

    Science.gov (United States)

    Lavin, Martin F; Kozlov, Sergei; Gatei, Magtouf; Kijas, Amanda W

    2015-10-23

    The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.

  9. The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

    Science.gov (United States)

    Xu, Ye; Sun, Yingli; Jiang, Xiaofeng; Ayrapetov, Marina K.; Moskwa, Patryk; Yang, Shenghong; Weinstock, David M.

    2010-01-01

    The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage. PMID:20876283

  10. SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Waaqo Daddacha

    2017-08-01

    Full Text Available DNA double-strand break (DSB repair by homologous recombination (HR is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.

  11. A sharp Pif1-dependent threshold separates DNA double-strand breaks from critically short telomeres

    NARCIS (Netherlands)

    Strecker, Jonathan; Stinus, Sonia; Caballero, Mariana Pliego; Szilard, Rachel K.; Chang, Michael; Durocher, Daniel

    2017-01-01

    DNA double-strand breaks (DSBs) and short telomeres are structurally similar, yet have diametrically opposed fates. Cells must repair DSBs while blocking the action of telomerase on these ends. Short telomeres must avoid recognition by the DNA damage response while promoting telomerase recruitment.

  12. Breaking the Rules in Style.

    Science.gov (United States)

    Romano, Tom

    1988-01-01

    Describes how allowing students to break the rules of standard writing can increase students' creativity in their written expression. Discusses several traits of this alternate style, or "Grammar B," including sentence fragments, double voice, lists, and spelling variations. (MM)

  13. Positive Disintegration as a Process of Symmetry Breaking.

    Science.gov (United States)

    Laycraft, Krystyna

    2017-04-01

    This article presents an analysis of the positive disintegration as a process of symmetry breaking. Symmetry breaking plays a major role in self-organized patterns formation and correlates directly to increasing complexity and function specialization. According to Dabrowski, a creator of the Theory of Positive Disintegration, the change from lower to higher levels of human development requires a major restructuring of an individual's psychological makeup. Each level of human development is a relatively stable and coherent configuration of emotional-cognitive patterns called developmental dynamisms. Their main function is to restructure a mental structure by breaking the symmetry of a low level and bringing differentiation and then integration to higher levels. The positive disintegration is then the process of transitions from a lower level of high symmetry and low complexity to higher levels of low symmetry and high complexity of mental structure.

  14. Hypospadias repair - discharge

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/patientinstructions/000158.htm Hypospadias repair - discharge To use the sharing features on this page, please enable JavaScript. Your child had hypospadias repair to fix a birth defect in which ...

  15. Anterior vaginal wall repair

    Science.gov (United States)

    ... may have you: Learn pelvic floor muscle exercises ( Kegel exercises ) Use estrogen cream in your vagina Try ... repair; Urinary incontinence - vaginal wall repair Patient Instructions Kegel exercises - self-care Self catheterization - female Suprapubic catheter ...

  16. Collision Repair Campaign

    Science.gov (United States)

    The Collision Repair Campaign targets meaningful risk reduction in the Collision Repair source category to reduce air toxic emissions in their communities. The Campaign also helps shops to work towards early compliance with the Auto Body Rule.

  17. Pectus excavatum repair

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/002949.htm Pectus excavatum repair To use the sharing features on this page, please enable JavaScript. Pectus excavatum repair is surgery to correct pectus excavatum . This ...

  18. Laparoscopic Inguinal Hernia Repair

    Science.gov (United States)

    ... Global Affairs and Humanitarian Efforts Log In Laparoscopic Inguinal Hernia Repair Surgery Patient Information from SAGES Download PDF Find a SAGES Surgeon About Your Inguinal Hernia and Laparoscopic Repair: Approximately 600,000 inguinal or ...

  19. Repair of DNA damaged by ionizing radiation and other oxidative agents in yeast and human

    Energy Technology Data Exchange (ETDEWEB)

    Louisek Prakash

    2000-01-15

    the carboxyl-terminus of yeast Apn2 protein does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl-terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo. We also carried out further biochemical characterization of the yeast Apn2 protein. As mentioned above, oxidative DNA damaging agents, such as hydrogen peroxide, produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. We found that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'5' exonuclease activities, and mutation of the active site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, our genetic studies indicate the involvement of APN2 in the repair of hydrogen peroxide induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we have concluded that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species. Other studies from our laboratory indicate that the yeast APN1 and APN2 genes provide alternate pathways for the repair of abasic sites and for the repair of single strand breaks with 3'-blocked termini. The apn1 deletion apn2 deletion mutant is highly sensitive to both the alkylating agent methyl methanesulfonate and to the oxidizing agent hydrogen peroxide. While the apn1 deletion and apn2 deletion single mutants are proficient in repairing single strand breaks arising in DNA following treatment with hydrogen peroxide, the repair of abasic sites as well as of single strand DNA breaks with 3'-blocked termini is greatly reduced in

  20. Repair of DNA damaged by ionizing radiation and other oxidative agents in yeast and human

    Energy Technology Data Exchange (ETDEWEB)

    Louise Prakash

    2000-01-15

    carboxyl-terminus of yeast Apn2 protein does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl-terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo. We also carried out further biochemical characterization of the yeast Apn2 protein. As mentioned above, oxidative DNA damaging agents, such as hydrogen peroxide, produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. We found that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'5' exonuclease activities, and mutation of the active site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, our genetic studies indicate the involvement of APN2 in the repair of hydrogen peroxide induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we have concluded that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species. Other studies from our laboratory indicate that the yeast APN1 and APN2 genes provide alternate pathways for the repair of abasic sites and for the repair of single strand breaks with 3'-blocked termini. The apn1 deletion apn2 deletion mutant is highly sensitive to both the alkylating agent methyl methanesulfonate and to the oxidizing agent hydrogen peroxide. While the apn1 deletion and apn2 deletion single mutants are proficient in repairing single strand breaks arising in DNA following treatment with hydrogen peroxide, the repair of abasic sites as well as of single strand DNA breaks with 3'-blocked termini is greatly reduced in the apn1

  1. Exploration of methods to identify polymorphisms associated with variation in DNA repair capacity phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Jones, I M; Thomas, C B; Xi, T; Mohrenweiser, H W; Nelson, D O

    2006-07-03

    Elucidating the relationship between polymorphic sequences and risk of common disease is a challenge. For example, although it is clear that variation in DNA repair genes is associated with familial cancer, aging and neurological disease, progress toward identifying polymorphisms associated with elevated risk of sporadic disease has been slow. This is partly due to the complexity of the genetic variation, the existence of large numbers of mostly low frequency variants and the contribution of many genes to variation in susceptibility. There has been limited development of methods to find associations between genotypes having many polymorphisms and pathway function or health outcome. We have explored several statistical methods for identifying polymorphisms associated with variation in DNA repair phenotypes. The model system used was 80 cell lines that had been resequenced to identify variation; 191 single nucleotide substitution polymorphisms (SNPs) are included, of which 172 are in 31 base excision repair pathway genes, 19 in 5 anti-oxidation genes, and DNA repair phenotypes based on single strand breaks measured by the alkaline Comet assay. Univariate analyses were of limited value in identifying SNPs associated with phenotype variation. Of the multivariable model selection methods tested: the easiest that provided reduced error of prediction of phenotype was simple counting of the variant alleles predicted to encode proteins with reduced activity, which led to a genotype including 52 SNPs; the best and most parsimonious model was achieved using a two-step analysis without regard to potential functional relevance: first SNPs were ranked by importance determined by Random Forests Regression (RFR), followed by cross-validation in a second round of RFR modeling that included ever more SNPs in declining order of importance. With this approach 6 SNPs were found to minimize prediction error. The results should encourage research into utilization of multivariate

  2. Architecture of the Pol III–clamp–exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair

    Science.gov (United States)

    Toste Rêgo, Ana; Holding, Andrew N; Kent, Helen; Lamers, Meindert H

    2013-01-01

    DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ɛ that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III–clamp–exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis. PMID:23549287

  3. Breaking Barriers in Polymer Additive Manufacturing

    Energy Technology Data Exchange (ETDEWEB)

    Love, Lonnie J [ORNL; Duty, Chad E [ORNL; Post, Brian K [ORNL; Lind, Randall F [ORNL; Lloyd, Peter D [ORNL; Kunc, Vlastimil [ORNL; Peter, William H [ORNL; Blue, Craig A [ORNL

    2015-01-01

    Additive Manufacturing (AM) enables the creation of complex structures directly from a computer-aided design (CAD). There are limitations that prevent the technology from realizing its full potential. AM has been criticized for being slow and expensive with limited build size. Oak Ridge National Laboratory (ORNL) has developed a large scale AM system that improves upon each of these areas by more than an order of magnitude. The Big Area Additive Manufacturing (BAAM) system directly converts low cost pellets into a large, three-dimensional part at a rate exceeding 25 kg/h. By breaking these traditional barriers, it is possible for polymer AM to penetrate new manufacturing markets.

  4. A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chakraborty, Ujani; George, Carolyn M; Lyndaker, Amy M; Alani, Eric

    2016-02-01

    Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker's yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker's yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 3' tails. Thus 3' tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 3' tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability. Copyright © 2016 by the Genetics Society of America.

  5. Ideas that break through

    CERN Multimedia

    Antonella Del Rosso

    2013-01-01

    The EU-cofunded project ULICE (Union of Light Ion Centres in Europe) was launched in 2009 in response to the need to share clinical experience in hadron therapy treatment in Europe and knowledge of the associated complex technical aspects. After four successful years of activity the project is now over but the “transnational access” idea will survive thanks to an extension granted by the European Commission.   A treatment room at CNAO, the Italian centre for hadron therapy. CNAO is participating in ULICE’s transnational access initiative. Image: CNAO. Until a few years ago, the landscape of hadron therapy in Europe was advancing in a fragmented way and facilities were being built without a common shared approach. EU-cofunded projects such as ENLIGHT, ULICE, PARTNER, ENVISION and ENTERVISION helped to build a unified platform where the different – private and public – stakeholders were able to share their views and practical experience in the ...

  6. The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans.

    Science.gov (United States)

    Meier, Bettina; Barber, Louise J; Liu, Yan; Shtessel, Ludmila; Boulton, Simon J; Gartner, Anton; Ahmed, Shawn

    2009-11-18

    The telomerase reverse transcriptase adds de novo DNA repeats to chromosome termini. Here we define Caenorhabditis elegans MRT-1 as a novel factor required for telomerase-mediated telomere replication and the DNA-damage response. MRT-1 is composed of an N-terminal domain homologous to the second OB-fold of POT1 telomere-binding proteins and a C-terminal SNM1 family nuclease domain, which confer single-strand DNA-binding and processive 3'-to-5' exonuclease activity, respectively. Furthermore, telomerase activity in vivo depends on a functional MRT-1 OB-fold. We show that MRT-1 acts in the same telomere replication pathway as telomerase and the 9-1-1 DNA-damage response complex. MRT-1 is dispensable for DNA double-strand break repair, but functions with the 9-1-1 complex to promote DNA interstrand cross-link (ICL) repair. Our data reveal MRT-1 as a dual-domain protein required for telomerase function and ICL repair, which raises the possibility that telomeres and ICL lesions may share a common feature that plays a critical role in de novo telomere repeat addition.

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

    Science.gov (United States)

    Park, Sojin; Choi, Seoyun; Ahn, Byungchan

    2016-01-01

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

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

    Science.gov (United States)

    Park, Sojin; Choi, Seoyun; Ahn, Byungchan

    2016-03-01

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

  9. On breaks of the Indian monsoon

    Indian Academy of Sciences (India)

    Further, there are three or four active-break cycles in a season according to Webster et al (1998) which implies a time scale of about 40 days for which Goswami and Mohan (2000), and Annamalai and Slingo (2001) have studied breaks and active minus break fluctuations. On the other hand, neither the traditional breaks ...

  10. Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Ifigeneia V. Mavragani

    2017-07-01

    Full Text Available Cellular effects of ionizing radiation (IR are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs, single strand breaks (SSBs and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1 repair resistant, increasing genomic instability (GI and malignant transformation and (2 can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity. Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair.

  11. DNA repair: Dynamic defenders against cancer and aging

    Energy Technology Data Exchange (ETDEWEB)

    Fuss, Jill O.; Cooper, Priscilla K.

    2006-04-01

    (UV) component of sunlight. NER can be divided into two classes based on where the repair occurs. NER occurring in DNA that is not undergoing transcription (i.e., most of the genome) is called global genome repair (GGR or GGNER), while NER taking place in the transcribed strand of active genes is called transcription-coupled repair (TCR or TC-NER). We will explore NER in more detail below. Mismatch repair (MMR) is another type of excision repair that specifically removes mispaired bases resulting from replication errors. DNA damage can also result in breaks in the DNA backbone, in one or both strands. Single-strand breaks (SSBs) are efficiently repaired by a mechanism that shares common features with the later steps in BER. Double-strand breaks (DSBs) are especially devastating since by definition there is no intact complementary strand to serve as a template for repair, and even one unrepaired DSB can be lethal [3]. In cells that have replicated their DNA prior to cell division, the missing information can be supplied by the duplicate copy, or sister chromatid, and DSBs in these cells are faithfully repaired by homologous recombination involving the exchange of strands of DNA between the two copies. However, most cells in the body are non-dividing, and in these cells the major mechanism for repairing DSBs is by non-homologous end joining (NHEJ), which as the name implies involves joining two broken DNA ends together without a requirement for homologous sequence and which therefore has a high potential for loss of genetic information.

  12. Blinking and tear break-up during four visual tasks.

    Science.gov (United States)

    Himebaugh, Nikole L; Begley, Carolyn G; Bradley, Arthur; Wilkinson, Jenni A

    2009-02-01

    This study investigates the relationship between blinking, tear film break-up, and ocular symptoms for normal and dry eye subjects performing four different visual tasks. Sixteen control and sixteen dry eye subjects performed four visual tasks (looking straight ahead, watching a movie, identifying rapidly changing letters, and playing a computer game) while blink patterns and fluorescein images of the tear film were videotaped. Pre and posttesting symptom questionnaires, querying the intensity of nine symptoms of ocular irritation, were completed by all subjects. Blink rate and blink amplitude were computed from digitized videos. The percentage of tear film break-up before the blink was calculated. Dry eye subjects had a significantly higher blink rate (p = 0.017, t-test). Both groups blinked significantly less during the game and letter tasks (p break-up in normal subjects was typically inferior; whereas dry eye subjects showed more tear break-up centrally and superiorly. Real-time video recording of tear break-up and blink behavior pointed to complex interaction between the two. Dry eye subjects shifted more toward intense ocular symptoms at posttesting (p break-up during normal visual tasks may explain the increased level of ocular discomfort symptoms reported at the end of the day, particularly in dry eye patients.

  13. Isospin breaking from diquark clustering

    Science.gov (United States)

    Gibbs, W. R.; Dedonder, Jean-Pierre

    2017-09-01

    Background: Although SU(2) isospin symmetry is generally assumed in the basic theory of the strong interaction, a number of significant violations have been observed in scattering and bound states of nucleons. Many of these violations can be attributed to the electromagnetic interaction but the question of how much of the violation is due to it remains open. Purpose: To establish the connection between diquark clustering in the two-nucleon system and isospin breaking from the Coulomb interaction between the members of diquark pairs. Method: A schematic model based on clustering of quarks in the interior of the confinement region of the two-nucleon system is introduced and evaluated. In this model the Coulomb interaction is the source of all isospin breaking. It draws on a picture of the quark density based on the diquark-quark model of hadron structure which has been investigated by a number of groups. Results: The model produces three isospin breaking potentials connecting the unbroken value of the low-energy scattering amplitude to those of the p p , n n , and n p singlet channels. A simple test of the potentials in the three-nucleon energy difference problem yields results in agreement with the known binding energy difference. Conclusion: The illustrative model suggests that the breaking seen in the low-energy nucleon-nucleon (NN) interaction may be understood in terms of the Coulomb force between members of diquark clusters. It allows the prediction of the charge symmetry breaking interaction and the n n scattering length from the well measured n p singlet scattering length. Values of the n n scattering length around -18 fm are favored. Since the model is based on the quark picture, it can be easily extended, in the SU(3) limit, to calculate isospin breaking in the strange sector in the corresponding channels. A natural consequence of isospin breaking from diquark clustering is that the breaking in the strange sector, as measured by the separation energy

  14. Hand function after nerve repair.

    Science.gov (United States)

    Lundborg, G; Rosén, B

    2007-02-01

    Treatment of injuries to major nerve trunks in the hand and upper extremity remains a major and challenging reconstructive problem. Such injuries may cause long-lasting disabilities in terms of lost fine sensory and motor functions. Nowadays there is no surgical repair technique that can ensure recovery of tactile discrimination in the hand of an adult patient following nerve repair while very young individuals usually regain a complete recovery of functional sensibility. Post-traumatic nerve regeneration is a complex biological process where the outcome depends on multiple biological and environmental factors such as survival of nerve cells, axonal regeneration rate, extent of axonal misdirection, type of injury, type of nerve, level of the lesion, age of the patient and compliance to training. A major problem is the cortical functional reorganization of hand representation which occurs as a result of axonal misdirection. Although protective sensibility usually occurs following nerve repair, tactile discriminative functions seldom recover--a direct result of cortical remapping. Sensory re-education programmes are routinely applied to facilitate understanding of the new sensory patterns provided by the hand. New trends in hand rehabilitation focus on modulation of central nervous processes rather than peripheral factors. Principles are being evolved to maintain the cortical hand representation by using the brain capacity for visuo-tactile and audio-tactile interaction for the initial phase following nerve injury and repair (phase 1). After the start of the re-innervation of the hand (phase 2), selective de-afferentation, such as cutaneous anaesthesia of the forearm of the injured hand, allows expansion of the nerve-injured cortical hand representation, thereby enhancing the effects of sensory relearning. Recent data support the view that training protocols specifically addressing the relearning process substantially increase the possibilities for improved

  15. 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.

  16. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

    2007-02-07

    The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

  17. Shuttle Repair Tools Automate Vehicle Maintenance

    Science.gov (United States)

    2013-01-01

    Successfully building, flying, and maintaining the space shuttles was an immensely complex job that required a high level of detailed, precise engineering. After each shuttle landed, it entered a maintenance, repair, and overhaul (MRO) phase. Each system was thoroughly checked and tested, and worn or damaged parts replaced, before the shuttle was rolled out for its next mission. During the MRO period, workers needed to record exactly what needed replacing and why, as well as follow precise guidelines and procedures in making their repairs. That meant traceability, and with it lots of paperwork. In 2007, the number of reports generated during electrical system repairs was getting out of hand-placing among the top three systems in terms of paperwork volume. Repair specialists at Kennedy Space Center were unhappy spending so much time at a desk and so little time actually working on the shuttle. "Engineers weren't spending their time doing technical work," says Joseph Schuh, an electrical engineer at Kennedy. "Instead, they were busy with repetitive, time-consuming processes that, while important in their own right, provided a low return on time invested." The strain of such inefficiency was bad enough that slow electrical repairs jeopardized rollout on several occasions. Knowing there had to be a way to streamline operations, Kennedy asked Martin Belson, a project manager with 30 years experience as an aerospace contractor, to co-lead a team in developing software that would reduce the effort required to document shuttle repairs. The result was System Maintenance Automated Repair Tasks (SMART) software. SMART is a tool for aggregating and applying information on every aspect of repairs, from procedures and instructions to a vehicle s troubleshooting history. Drawing on that data, SMART largely automates the processes of generating repair instructions and post-repair paperwork. In the case of the space shuttle, this meant that SMART had 30 years worth of operations

  18. Contemporary Concepts for the Bilateral Cleft Lip and Nasal Repair

    OpenAIRE

    Khosla, Rohit K.; McGregor, Jyoti; Kelley, Patrick K.; Gruss, Joseph S.

    2012-01-01

    The bilateral cleft lip and nasal deformity presents a complex challenge for repair. Surgical techniques continue to evolve and are focused on primary anatomic realignment of the tissues. This can be accomplished in a single-stage or two-stage repair early in infancy to provide a foundation for future growth of the lip and nasal tissue. Most cleft surgeons currently perform a single-stage repair for simplifying patient care. Certain institutions utilize presurgical orthopedics for alignment o...

  19. supersymmetry breaking with extra dimensions

    Indian Academy of Sciences (India)

    physics pp. 497-511 supersymmetry breaking with extra dimensions. FABIO ZWIRNER. Theory Division, CERN, CH-1211 Geneva 23, Switzerland. On leave from: Physics ... theory which accounts for all the observed interactions at the presently available ... For some standard reviews of sUsY and of the MssM, with lists.

  20. Aluminum break-point contacts

    NARCIS (Netherlands)

    Heinemann, Martina; Groot, R.A. de

    1997-01-01

    Ab initio molecular dynamics is used to study the contribution of a single Al atom to an aluminum breakpoint contact during the final stages of breaking and the initial stages of the formation of such a contact. A hysteresis effect is found in excellent agreement with experiment and the form of the

  1. Breaking Carbon Lock-in

    DEFF Research Database (Denmark)

    Driscoll, Patrick Arthur

    2014-01-01

    This central focus of this paper is to highlight the ways in which path dependencies and increasing returns (network effects) serve to reinforce carbon lock-in in large-scale road transportation infrastructure projects. Breaking carbon lock-in requires drastic changes in the way we plan future...

  2. Baryon and chiral symmetry breaking

    Energy Technology Data Exchange (ETDEWEB)

    Gorsky, A. [Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia and Moscow Institute of Physics and Technology (MIPT), Dolgoprudny (Russian Federation); Krikun, A. [NORDITA, KTH Royal Institute of Technology and Stockholm University Stockholm, Sweden and Institute for Theoretical and Experimental Physics (ITEP), Moscow (Russian Federation)

    2014-07-23

    We briefly review the generalized Skyrmion model for the baryon recently suggested by us. It takes into account the tower of vector and axial mesons as well as the chiral symmetry breaking. The generalized Skyrmion model provides the qualitative explanation of the Ioffe’s formula for the baryon mass.

  3. Breaking Rules–Making Bonds

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 21; Issue 1. Breaking Rules - Making Bonds. A G Samuelson. General Article Volume 21 Issue 1 January 2016 pp 43-48. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/021/01/0043-0048. Keywords.

  4. Small Break Air Ingress Experiment

    Energy Technology Data Exchange (ETDEWEB)

    Chang Oh; Eung Soo Kim

    2011-09-01

    The small break air-ingress experiment, described in this report, is designed to investigate air-ingress phenomena postulated to occur in pipes in a very high temperature gas-cooled reactor (VHTRs). During this experiment, air-ingress rates were measured for various flow and break conditions through small holes drilled into a pipe of the experimental apparatus. The holes were drilled at right angles to the pipe wall such that a direction vector drawn from the pipe centerline to the center of each hole was at right angles with respect to the pipe centerline. Thus the orientation of each hole was obtained by measuring the included angle between the direction vector of each hole with respect to a reference line anchored on the pipe centerline and pointing in the direction of the gravitational force. Using this reference system, the influence of several important parameters on the air ingress flow rate were measured including break orientation, break size, and flow velocity . The approach used to study the influence of these parameters on air ingress is based on measuring the changes in oxygen concentrations at various locations in the helium flow circulation system as a function of time using oxygen sensors (or detectors) to estimate the air-ingress rates through the holes. The test-section is constructed of a stainless steel pipe which had small holes drilled at the desired locations.

  5. Sediment transport under breaking waves

    DEFF Research Database (Denmark)

    Christensen, Erik Damgaard; Hjelmager Jensen, Jacob; Mayer, Stefan

    2000-01-01

    generated at the surface where the wave breaks as well as the turbulence generated near the bed due to the wave-motion and the undertow. In general, the levels of turbulent kinetic energy are found to be higher than experiments show. This results in an over prediction of the sediment transport. Nevertheless...

  6. Strong coupling electroweak symmetry breaking

    Energy Technology Data Exchange (ETDEWEB)

    Barklow, T.L. [Stanford Linear Accelerator Center, Menlo Park, CA (United States); Burdman, G. [Univ. of Wisconsin, Madison, WI (United States). Dept. of Physics; Chivukula, R.S. [Boston Univ., MA (United States). Dept. of Physics

    1997-04-01

    The authors review models of electroweak symmetry breaking due to new strong interactions at the TeV energy scale and discuss the prospects for their experimental tests. They emphasize the direct observation of the new interactions through high-energy scattering of vector bosons. They also discuss indirect probes of the new interactions and exotic particles predicted by specific theoretical models.

  7. Do DNA Double-Strand Breaks Drive Aging?

    Science.gov (United States)

    White, Ryan R; Vijg, Jan

    2016-09-01

    DNA double-strand breaks (DSBs) are rare, but highly toxic, lesions requiring orchestrated and conserved machinery to prevent adverse consequences, such as cell death and cancer-causing genome structural mutations. DSBs trigger the DNA damage response (DDR) that directs a cell to repair the break, undergo apoptosis, or become senescent. There is increasing evidence that the various endpoints of DSB processing by different cells and tissues are part of the aging phenotype, with each stage of the DDR associated with specific aging pathologies. In this Perspective, we discuss the possibility that DSBs are major drivers of intrinsic aging, highlighting the dynamics of spontaneous DSBs in relation to aging, the distinct age-related pathologies induced by DSBs, and the segmental progeroid phenotypes in humans and mice with genetic defects in DSB repair. A model is presented as to how DSBs could drive some of the basic mechanisms underlying age-related functional decline and death. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Entropy in DNA Double-Strand Break, Detection and Signaling

    Science.gov (United States)

    Zhang, Yang; Schindler, Christina; Heermann, Dieter

    2014-03-01

    In biology, the term entropy is often understood as a measure of disorder - a restrictive interpretation that can even be misleading. Recently it has become clearer and clearer that entropy, contrary to conventional wisdom, can help to order and guide biological processes in living cells. DNA double-strand breaks (DSBs) are among the most dangerous lesions and efficient damage detection and repair is essential for organism viability. However, what remains unknown is the precise mechanism of targeting the site of damage within billions of intact nucleotides and a crowded nuclear environment, a process which is often referred to as recruitment or signaling. Here we show that the change in entropy associated with inflicting a DSB facilitates the recruitment of damage sensor proteins. By means of computational modeling we found that higher mobility and local chromatin structure accelerate protein association at DSB ends. We compared the effect of different chromatin architectures on protein dynamics and concentrations in the vicinity of DSBs, and related these results to experiments on repair in heterochromatin. Our results demonstrate how entropy contributes to a more efficient damage detection. We identify entropy as the physical basis for DNA double-strand break signaling.

  9. Dose response of gamma rays and iron nuclei for induction of chromosomal aberrations in normal and repair-deficient cell lines.

    Science.gov (United States)

    George, Kerry A; Hada, Megumi; Jackson, Lori J; Elliott, Todd; Kawata, Tetsuya; Pluth, Janice M; Cucinotta, Francis A

    2009-06-01

    We studied the effects of DNA double-strand break (DSB) repair deficiencies on chromosomal aberration frequency using low doses (gamma rays and high-energy iron ions (LET = 151 keV/microm). Chromosomal aberrations were measured using the fluorescence whole-chromosome painting technique. The cell lines included fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome) and gliomablastoma cells proficient in or lacking DNA-dependent protein kinase (DNA-PK) activity. The yields of both simple and complex chromosomal aberrations were increased in DSB repair-defective cells compared to normal cells; the increase was more than twofold higher for gamma rays compared to iron nuclei. For gamma-ray-induced aberrations, the ATM- and NBS-defective lines were found to have significantly larger quadratic components compared to normal fibroblasts for both simple and complex aberrations, while the linear dose-response term was significantly higher only for the NBS cells. For simple and complex aberrations induced by iron nuclei, regression models preferred purely linear and quadratic dose responses, respectively, for each cell line studied. RBEs were reduced relative to normal cells for all of the DSB repair-defective lines, with the DNA-PK-deficient cells found to have RBEs near unity. The large increase in the quadratic dose-response terms in the DSB repair-deficient cell lines points to the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and to minimize aberration formation. The differences found between AT and NBS cells at lower doses suggest important questions about the applicability of observations of radiation sensitivity at high doses to low-dose exposures.

  10. Suture-Only Repair Versus Suture Anchor–Augmented Repair for Achilles Tendon Ruptures With a Short Distal Stump

    Science.gov (United States)

    Boin, Michael A.; Dorweiler, Matthew A.; McMellen, Christopher J.; Gould, Gregory C.; Laughlin, Richard T.

    2017-01-01

    Background: Chronic noninsertional Achilles tendinosis can result in an acute Achilles tendon rupture with a short distal stump. In such tendon ruptures, there is a limited amount of adequate tissue that can hold suture, thus presenting a challenge for surgeons who elect to treat the rupture operatively. Hypothesis: Adding suture anchors to the repair construct may result in biomechanically stronger repairs compared with a suture-only technique. Study Design: Controlled laboratory study. Methods: Nine paired Achilles-calcaneus complexes were harvested from cadavers. An artificial Achilles rupture was created 2 cm proximal to the insertion on the calcaneus. One specimen from each cadaver was assigned to a suture-only or a suture anchor–augmented repair. The contralateral specimen of the same cadaver received the opposing repair. Cyclic testing was then performed at 10 to 100 N for 2000 cycles, and load-to-failure testing was performed at 0.2 mm/s. This was followed by analysis of repair displacement, gapping at repair site, peak load to failure, and failure mode. Results: The suture anchor–augmented repair exhibited a 116% lower displacement compared with the suture-only repair (mean ± SD, 1.54 ± 1.13 vs 3.33 ± 1.47 mm, respectively; P suture anchor–augmented repair also exhibited a 45% greater load to failure compared with the suture-only repair (303.50 ± 102.81 vs 209.09 ± 48.12 N, respectively; P Suture anchor–augmented repairs performed on acute Achilles tendon ruptures with a short distal stump are biomechanically stronger than suture-only repairs. Clinical Relevance: Our results support the use of suture anchor–augmented repairs for a biomechanically stronger construct in Achilles tendon ruptures with a short distal stump. Biomechanically stronger repairs may lead to less tendon repair gapping and failure, increasing the ability to start early active rehabilitation protocols and thus improving patient outcomes. PMID:28203592

  11. Assessing the effect of a fuel break network to reduce burnt area and wildfire risk transmission

    Science.gov (United States)

    Tiago M. Oliveira; Ana M. G. Barros; Alan A. Ager; Paulo M. Fernandes

    2016-01-01

    Wildfires pose complex challenges to policymakers and fire agencies. Fuel break networks and area-wide fuel treatments are risk-management options to reduce losses from large fires. Two fuel management scenarios covering 3% of the fire-prone Algarve region of Portugal and differing in the intensity of treatment in 120-m wide fuel breaks were examined and compared with...

  12. A model of intrinsic symmetry breaking

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Li [Research Center for Quantum Manipulation, Department of Physics, Fudan University, Shanghai 200433 (China); Li, Sheng [Department of Physics, Zhejiang Normal University, Zhejiang 310004 (China); George, Thomas F., E-mail: tfgeorge@umsl.edu [Office of the Chancellor and Center for Nanoscience, Department of Chemistry and Biochemistry, University of Missouri-St. Louis, St. Louis, MO 63121 (United States); Department of Physics and Astronomy, University of Missouri-St. Louis, St. Louis, MO 63121 (United States); Sun, Xin, E-mail: xin_sun@fudan.edu.cn [Research Center for Quantum Manipulation, Department of Physics, Fudan University, Shanghai 200433 (China)

    2013-11-01

    Different from the symmetry breaking associated with a phase transition, which occurs when the controlling parameter is manipulated across a critical point, the sym