RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

Science.gov (United States)

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication.

Science.gov (United States)

Morrow, Carl A; Nguyen, Michael O; Fower, Andrew; Wong, Io Nam; Osman, Fekret; Bryer, Claire; Whitby, Matthew C

2017-06-06

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.

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.

FANCJ couples replication past natural fork barriers with maintenance of chromatin structure.

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Schwab, Rebekka A; Nieminuszczy, Jadwiga; Shin-ya, Kazuo; Niedzwiedz, Wojciech

2013-04-01

Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer-predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner.

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

Science.gov (United States)

Schalbetter, Stephanie A; Mansoubi, Sahar; Chambers, Anna L; Downs, Jessica A; Baxter, Jonathan

2015-08-18

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

The progression of replication forks at natural replication barriers in live bacteria.

Science.gov (United States)

Moolman, M Charl; Tiruvadi Krishnan, Sriram; Kerssemakers, Jacob W J; de Leeuw, Roy; Lorent, Vincent; Sherratt, David J; Dekker, Nynke H

2016-07-27

Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks Differently

Science.gov (United States)

Yu, Chuanhe; Gan, Haiyun

2017-01-01

ABSTRACT Three DNA polymerases, polymerases α, δ, and ε (Pol α, Pol δ, and Pol ε), are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has a distinct pattern of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has greater contact with the nascent single-stranded DNA (ssDNA) of the leading strand on active forks than on stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitates the resumption of DNA synthesis after stress removal. PMID:28784720

The progression of replication forks at natural replication barriers in live bacteria

NARCIS (Netherlands)

Moolman, M.C.; Tiruvadi Krishnan, S; Kerssemakers, J.W.J.; de Leeuw, R.; Lorent, V.J.F.; Sherratt, David J.; Dekker, N.H.

2016-01-01

Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

Science.gov (United States)

Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

2017-01-05

The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The fork and the kinase: a DNA replication tale from a CHK1 perspective.

Science.gov (United States)

González Besteiro, Marina A; Gottifredi, Vanesa

2015-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. Checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged DNA. Subsequently, Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Indeed, such findings have unveiled a puzzling connection between Chk1 and DNA lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, Chk1 is a multifaceted and versatile signaling factor that acts at ongoing forks and replication origins to determine the extent and quality of the cellular response to replication stress. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell lethality after selective irradiation of the DNA replication fork

International Nuclear Information System (INIS)

Hofer, K.G.; Warters, R.L.

1985-01-01

It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125 I-iododeoxyuridine ( 125 IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 μg/ml) reduced cellular 125 IUdR incorporation to 3-5% of the control value. The residual 125 I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125 I decays at the replication fork to that of 125 I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D 0 : 101 125 I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome. (orig.)

Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

KAUST Repository

Hamdan, Samir

2010-04-09

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field. 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

KAUST Repository

Hamdan, Samir; van Oijen, Antoine M.

2010-01-01

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field. 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

Science.gov (United States)

Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

2017-09-15

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

DEFF Research Database (Denmark)

Larsen, Nicolai B; Sass, Ehud; Suski, Catherine

2014-01-01

Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus...... as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications....

Replication fork stability confers chemoresistance in BRCA-deficient cells

DEFF Research Database (Denmark)

Chaudhuri, Arnab Ray; Callen, Elsa; Ding, Xia

2016-01-01

/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11......Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3...... nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations...

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

Science.gov (United States)

Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S.

2016-01-01

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

Directory of Open Access Journals (Sweden)

Therese Wilhelm

2016-05-01

Full Text Available Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es. Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3% rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing, and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

Science.gov (United States)

Wilhelm, Therese; Ragu, Sandrine; Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S

2016-05-01

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response.

Science.gov (United States)

Mori, Tetsuya; Nakamura, Tatsuro; Okazaki, Naoto; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

2012-01-01

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

Molecular basis for PrimPol recruitment to replication forks by RPA.

Science.gov (United States)

Guilliam, Thomas A; Brissett, Nigel C; Ehlinger, Aaron; Keen, Benjamin A; Kolesar, Peter; Taylor, Elaine M; Bailey, Laura J; Lindsay, Howard D; Chazin, Walter J; Doherty, Aidan J

2017-05-23

DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

Directory of Open Access Journals (Sweden)

Manjula Pandey

2014-03-01

Full Text Available By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

Disruption of PCNA-lamins A/C interactions by prelamin A induces DNA replication fork stalling.

Science.gov (United States)

Cobb, Andrew M; Murray, Thomas V; Warren, Derek T; Liu, Yiwen; Shanahan, Catherine M

2016-09-02

The accumulation of prelamin A is linked to disruption of cellular homeostasis, tissue degeneration and aging. Its expression is implicated in compromised genome stability and increased levels of DNA damage, but to date there is no complete explanation for how prelamin A exerts its toxic effects. As the nuclear lamina is important for DNA replication we wanted to investigate the relationship between prelamin A expression and DNA replication fork stability. In this study we report that the expression of prelamin A in U2OS cells induced both mono-ubiquitination of proliferating cell nuclear antigen (PCNA) and subsequent induction of Pol η, two hallmarks of DNA replication fork stalling. Immunofluorescence microscopy revealed that cells expressing prelamin A presented with high levels of colocalisation between PCNA and γH2AX, indicating collapse of stalled DNA replication forks into DNA double-strand breaks. Subsequent protein-protein interaction assays showed prelamin A interacted with PCNA and that its presence mitigated interactions between PCNA and the mature nuclear lamina. Thus, we propose that the cytotoxicity of prelamin A arises in part, from it actively competing against mature lamin A to bind PCNA and that this destabilises DNA replication to induce fork stalling which in turn contributes to genomic instability.

Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

Science.gov (United States)

Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

2018-01-01

DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

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

Directory of Open Access Journals (Sweden)

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.

Physical Interaction between Replication Protein A (RPA) and MRN: Involvement of RPA2 Phosphorylation and the N-terminus of RPA1

OpenAIRE

Oakley, Greg; Tillison, Kristin; Opiyo, Stephen; Glanzer, Jason; Horn, Jeffrey M.; Patrick, Steve M.

2009-01-01

Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2 and RPA3 subunits that binds to ssDNA with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11/RAD50/NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-dsDNA junctions or breaks and pr...

Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1.

Science.gov (United States)

Pryce, David W; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J

2009-03-24

DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion.

Stalled replication forks generate a distinct mutational signature in yeast

DEFF Research Database (Denmark)

Larsen, Nicolai B.; Liberti, Sascha E.; Vogel, Ivan

2017-01-01

Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication...... Escherichia coli Tus/Ter complex) engineered into the yeast genome. We demonstrate that transient stalling at this barrier induces a distinct pattern of genome rearrangements in the newly replicated region behind the stalled fork, which primarily consist of localized losses and duplications of DNA sequences....... These genetic alterations arise through the aberrant repair of a single-stranded DNA gap, in a process that is dependent on Exo1- and Shu1-dependent homologous recombination repair (HRR). Furthermore, aberrant processing of HRR intermediates, and elevated HRR-associated mutagenesis, is detectable in a yeast...

DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

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Hua, Brian L.; Orr-Weaver, Terry L.

2017-01-01

Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression. Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation. PMID:28874453

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

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Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

2016-07-26

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

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Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

2013-11-01

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast.

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Larsen, Nicolai B; Sass, Ehud; Suski, Catherine; Mankouri, Hocine W; Hickson, Ian D

2014-04-07

Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus, to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it is neither Tof1-dependent nor counteracted by the Rrm3 helicase. Although the yeast replisome can overcome RF pausing at Tus-Ter modules, this event triggers site-specific homologous recombination that requires the RecQ helicase, Sgs1, for its timely resolution. We propose that Tus-Ter can be utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications.

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition

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Gill, Martin R.; Harun, Siti Norain; Halder, Swagata; Boghozian, Ramon A.; Ramadan, Kristijan; Ahmad, Haslina; Vallis, Katherine A.

2016-08-01

Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells

DEFF Research Database (Denmark)

Ahuja, Akshay K.; Jodkowska, Karolina; Teloni, Federico

2016-01-01

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX...... these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork...... slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity....

Inhibition of DNA chain elongation in Chinese hamster cells by damage localized behind the replication fork

Energy Technology Data Exchange (ETDEWEB)

Ben-Hur, E [Israel Atomic Energy Commission, Beersheba. Nuclear Research Center-Negev; Hagan, M P [Armed Forces Radiobiology Research Inst., Bethesda, MD (USA)

1984-05-01

Chinese hamster fibroblasts were pulse labelled with 5-bromodeoxyuridine and exposed at time intervals (Tsub(i)) to near-ultraviolet (U.V.A.) light in the presence of a bisbenzimidazole derivative (Hoechst 33342). The sensitivity of the cells in terms of colony forming ability fluctuated depending on Tsub(i). Inhibition of DNA synthesis also depended on Tsub(i) and was maximal when Tsub(i)=O. Using the alkaline elution technique it was shown that the effect of a large dose of light was to inhibit both initiation and elongation of DNA chains. These effects were most pronounced for Tsub(i)=O. It is concluded that DNA damage in an active replicon can inhibit initiation of new replicons and that damage localized behind the replication fork can retard elongation of nascent DNA chains. This effect on chain elongation decreases with increased distance of the damage from the replication fork.

Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites.

Directory of Open Access Journals (Sweden)

Xiao P Peng

2018-01-01

Full Text Available Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA array. Each rDNA repeat contains a programmed replication fork barrier (RFB established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.

Forging Ahead through Darkness: PCNA, Still the Principal Conductor at the Replication Fork.

Science.gov (United States)

Choe, Katherine N; Moldovan, George-Lucian

2017-02-02

Proliferating cell nuclear antigen (PCNA) lies at the center of the faithful duplication of eukaryotic genomes. With its distinctive doughnut-shaped molecular structure, PCNA was originally studied for its role in stimulating DNA polymerases. However, we now know that PCNA does much more than promote processive DNA synthesis. Because of the complexity of the events involved, cellular DNA replication poses major threats to genomic integrity. Whatever predicament lies ahead for the replication fork, PCNA is there to orchestrate the events necessary to handle it. Through its many protein interactions and various post-translational modifications, PCNA has far-reaching impacts on a myriad of cellular functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Modeling inhomogeneous DNA replication kinetics.

Directory of Open Access Journals (Sweden)

Michel G Gauthier

Full Text Available In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

A microhomology-mediated break-induced replication model for the origin of human copy number variation.

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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.

Hydroxyurea-Induced Replication Stress

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Kenza Lahkim Bennani-Belhaj

2010-01-01

Full Text Available Bloom's syndrome (BS displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU, which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.

Modulation of mutagenesis in eukaryotes by DNA replication fork dynamics and quality of nucleotide pools

Science.gov (United States)

Waisertreiger, Irina S.-R.; Liston, Victoria G.; Menezes, Miriam R.; Kim, Hyun-Min; Lobachev, Kirill S.; Stepchenkova, Elena I.; Tahirov, Tahir H.; Rogozin, Igor B.; Pavlov, Youri. I.

2014-01-01

The rate of mutations in eukaryotes depends on a plethora of factors and is not immediately derived from the fidelity of DNA polymerases (Pols). Replication of chromosomes containing the anti-parallel strands of duplex DNA occurs through the copying of leading and lagging strand templates by a trio of Pols α, δ and ε, with the assistance of Pol ζ and Y-family Pols at difficult DNA template structures or sites of DNA damage. The parameters of the synthesis at a given location are dictated by the quality and quantity of nucleotides in the pools, replication fork architecture, transcription status, regulation of Pol switches, and structure of chromatin. The result of these transactions is a subject of survey and editing by DNA repair. PMID:23055184

Mechanisms of DNA replication termination.

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Dewar, James M; Walter, Johannes C

2017-08-01

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

A Network of Multi-Tasking Proteins at the DNA Replication Fork Preserves Genome Stability.

Directory of Open Access Journals (Sweden)

2005-12-01

Full Text Available To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.

Distinct functions of human RecQ helicases during DNA replication.

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Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

2017-06-01

DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA replication in ultraviolet light irradiated Chinese hamster cells: the nature of replicon inhibition and post-replication repair

International Nuclear Information System (INIS)

Doniger, J.

1978-01-01

DNA replication in ultraviolet light irradiated Chinese hamster cells was studied using techniques of DNA fiber autoradiography and alkaline sucrose sedimentation. Bidirectionally growing replicons were observed in the autoradiograms independent of the irradiation conditions. After a dose of 5 J/m 2 at 254 nm the rate of fork progression was the same as in unirradiated cells, while the rate of replication was reduced by 50%. After a dose of 10J/m 2 the rate of fork progression was reduced 40%, while the replication rate was only 25% of normal. Therefore, at low doses of ultraviolet light irradiation, the inhibition of DNA replication is due to reduction in the number of functioning replicons, while at higher doses the rate of fork progression is also slowed. Those replicons which no longer function after irradiation are blocked in fork movement rather than replicon initiation. After irradiation, pulse label was first incorporated into short nascent strands, the average size of which was approximately equal to the distance between pyrimidine dimers. Under conditions where post-replication repair occurs these short strands were eventually joined into larger pieces. Finally, the data show that slowing post-replication repair with caffeine does not slow fork movement. The results presented here support the post-replication repair model of 'gapped synthesis' and rule out a major role for 'replicative bypass'. (author)

Overcoming natural replication barriers: differential helicase requirements.

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Anand, Ranjith P; Shah, Kartik A; Niu, Hengyao; Sung, Patrick; Mirkin, Sergei M; Freudenreich, Catherine H

2012-02-01

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

Science.gov (United States)

Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

2016-06-01

A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Replisome speed determines the efficiency of the Tus−Ter replication termination barrier

KAUST Repository

Elshenawy, Mohamed; Jergic, Slobodan; Xu, Zhi Qiang; Sobhy, Mohamed Abdelmaboud; Takahashi, Masateru; Oakley, Aaron J.; Dixon, Nicholas E.; Hamdan, Samir

2015-01-01

In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes. ©2015 Macmillan Publishers Limited. All rights reserved.

Replisome speed determines the efficiency of the Tus−Ter replication termination barrier

KAUST Repository

Elshenawy, Mohamed

2015-08-31

In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a \\'replication fork trap\\' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes. ©2015 Macmillan Publishers Limited. All rights reserved.

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Kai, Mihoko; Wang, Teresa S.-F

2003-11-27

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Pol{kappa}). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

International Nuclear Information System (INIS)

Kai, Mihoko; Wang, Teresa S.-F.

2003-01-01

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polκ). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

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Toledo, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Claudia; Larsen, Dorthe Helena; Povlsen, Lou Klitgaard; Bekker-Jensen, Simon; Mailand, Niels; Bartek, Jiri; Lukas, Jiri

2013-11-21

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

Force regulated dynamics of RPA on a DNA fork.

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Kemmerich, Felix E; Daldrop, Peter; Pinto, Cosimo; Levikova, Maryna; Cejka, Petr; Seidel, Ralf

2016-07-08

Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg(2+) concentrations, such that human RPA can melt DNA in absence of force. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Insights into the Initiation of Eukaryotic DNA Replication.

Science.gov (United States)

Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

2015-01-01

The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

Directory of Open Access Journals (Sweden)

Audrey Costes

2010-12-01

Full Text Available We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.

Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus.

Science.gov (United States)

Duggin, Iain G; Dubarry, Nelly; Bell, Stephen D

2011-01-05

Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site. Sulfolobus encodes a single-Xer homologue and its deletion gave rise to cells with aberrant DNA contents and increased volumes. Identification of the chromosomal dif site that binds Xer in vivo, and biochemical characterization of Xer/dif recombination revealed that, in contrast to bacteria, dif is located outside the fork fusion zones. Therefore, it appears that replication termination and dimer resolution are temporally and spatially distinct processes in Sulfolobus.

DNA Replication Profiling Using Deep Sequencing.

Science.gov (United States)

Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

2018-01-01

Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

Science.gov (United States)

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

Science.gov (United States)

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

Physical interaction between replication protein A (RPA) and MRN: involvement of RPA2 phosphorylation and the N-terminus of RPA1.

Science.gov (United States)

Oakley, Greg G; Tillison, Kristin; Opiyo, Stephen A; Glanzer, Jason G; Horn, Jeffrey M; Patrick, Steve M

2009-08-11

Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2, and RPA3 subunits that binds to single-stranded DNA (ssDNA) with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11-RAD50-NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-double-stranded DNA (dsDNA) junctions or breaks, and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic weakens the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates interactions of RPA with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 shows that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage.

A novel class of mutations that affect DNA replication in E. coli

DEFF Research Database (Denmark)

Nordman, Jared; Skovgaard, Ole; Wright, Andrew

2007-01-01

Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the a...

Nonlinearity in superconductivity and Josephson junctions

International Nuclear Information System (INIS)

Lazarides, N.

1995-01-01

Within the framework of the Bardeen, Cooper and Schrieffers (BCS) theory, the influence of anisotropy on superconducting states are investigated. Crystal anisotropy exists in un-conventional low temperature superconductors as e.g. U 1-x Th x Be 13 and in high temperature superconductors. Starting from a phenomenological pairing interaction of the electrons or holes, the BCS approach is used to derive a set of coupled nonlinear algebraic equations for the momentum dependent gap parameter. The emphasis is put on bifurcation phenomena between s-, d-wave and mixed s- and d-wave symmetry and the influence on measurable quantities as the electron specific heat, spin susceptibility and Josephson tunnelling. Pitch-fork and perturbed pitch-fork bifurcations have been found separating s- and d-wave superconducting states from mixed s- and d-wave states. The additional superconducting states give rise to jumps in the electron specific heat below the transition temperature. These jumps are rounded in the case of perturbed pitch-fork bifurcations. An experiment to measure the sign of the interlayer interaction using dc SQUIDS is suggested. The Ambegaokar-Baratoff formalism has been used for calculating the quasiparticle current and the two phase coherent tunnelling currents in a Josephson junction made of anisotropic superconductors. It is shown that anisotropy can lead to a reduction in the product of the normal resistance and the critical current. For low voltages across the junction the usual resistively shunted Josephson model can be used. Finally, bunching in long circular Josephson junctions and suppression of chaos in point junctions have been investigated. (au) 113 refs

Exploiting replicative stress to treat cancer

DEFF Research Database (Denmark)

Dobbelstein, Matthias; Sørensen, Claus Storgaard

2015-01-01

DNA replication in cancer cells is accompanied by stalling and collapse of the replication fork and signalling in response to DNA damage and/or premature mitosis; these processes are collectively known as 'replicative stress'. Progress is being made to increase our understanding of the mechanisms...

From structure to mechanism—understanding initiation of DNA replication

Science.gov (United States)

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

2017-01-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

Managing Single-Stranded DNA during Replication Stress in Fission Yeast

Directory of Open Access Journals (Sweden)

Sarah A. Sabatinos

2015-09-01

Full Text Available Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA

DEFF Research Database (Denmark)

Toledo Lazaro, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt

2013-01-01

origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing...... induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells...

From structure to mechanism-understanding initiation of DNA replication.

Science.gov (United States)

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

2017-06-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

KAUST Repository

Lengsfeld, Bettina M.; Berry, Kayla N.; Ghosh, Sharmistha; Takahashi, Masateru; Francis, Nicole J.

2012-01-01

Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

KAUST Repository

Lengsfeld, Bettina M.

2012-09-17

Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

DEFF Research Database (Denmark)

Syed, Salahuddin; Madsen, Claus Desler; Rasmussen, Lene J.

2016-01-01

hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5...

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation.

Science.gov (United States)

Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

2018-01-01

Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

DEFF Research Database (Denmark)

Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

2018-01-01

" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication......Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien......-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types....

Replication Catastrophe

DEFF Research Database (Denmark)

Toledo, Luis; Neelsen, Kai John; Lukas, Jiri

2017-01-01

Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA...... breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication...... checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under...

Replication Stalling and Heteroduplex Formation within CAG/CTG Trinucleotide Repeats by Mismatch Repair

KAUST Repository

Viterbo, David; Michoud, Gregoire; Mosbach, Valentine; Dujon, Bernard; Richard, Guy-Franck

2016-01-01

Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

Replication Stalling and Heteroduplex Formation within CAG/CTG Trinucleotide Repeats by Mismatch Repair

KAUST Repository

Viterbo, David

2016-03-16

Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

RPA Mediates Recruitment of MRX to Forks and Double-Strand Breaks to Hold Sister Chromatids Together.

Science.gov (United States)

Seeber, Andrew; Hegnauer, Anna Maria; Hustedt, Nicole; Deshpande, Ishan; Poli, Jérôme; Eglinger, Jan; Pasero, Philippe; Gut, Heinz; Shinohara, Miki; Hopfner, Karl-Peter; Shimada, Kenji; Gasser, Susan M

2016-12-01

The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks. Copyright © 2016 Elsevier Inc. All rights reserved.

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

Science.gov (United States)

Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

Science.gov (United States)

Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

1994-07-01

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

NARCIS (Netherlands)

Tanner, Nathan A.; Loparo, Joseph J.; Oijen, Antoine M. van

2009-01-01

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides. The

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

Science.gov (United States)

García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

2016-01-01

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

Science.gov (United States)

van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J

1998-06-01

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

Science.gov (United States)

Mendez-Bermudez, Aaron; Lototska, Liudmyla; Bauwens, Serge; Giraud-Panis, Marie-Josèphe; Croce, Olivier; Jamet, Karine; Irizar, Agurtzane; Mowinckel, Macarena; Koundrioukoff, Stephane; Nottet, Nicolas; Almouzni, Genevieve; Teulade-Fichou, Mare-Paule; Schertzer, Michael; Perderiset, Mylène; Londoño-Vallejo, Arturo; Debatisse, Michelle; Gilson, Eric; Ye, Jing

2018-05-03

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

FANCD2 Maintains Fork Stability in BRCA1/2-Deficient Tumors and Promotes Alternative End-Joining DNA Repair

Directory of Open Access Journals (Sweden)

Zeina Kais

2016-06-01

Full Text Available BRCA1/2 proteins function in homologous recombination (HR-mediated DNA repair and cooperate with Fanconi anemia (FA proteins to maintain genomic integrity through replication fork stabilization. Loss of BRCA1/2 proteins results in DNA repair deficiency and replicative stress, leading to genomic instability and enhanced sensitivity to DNA-damaging agents. Recent studies have shown that BRCA1/2-deficient tumors upregulate Polθ-mediated alternative end-joining (alt-EJ repair as a survival mechanism. Whether other mechanisms maintain genomic integrity upon loss of BRCA1/2 proteins is currently unknown. Here we show that BRCA1/2-deficient tumors also upregulate FANCD2 activity. FANCD2 is required for fork protection and fork restart in BRCA1/2-deficient tumors. Moreover, FANCD2 promotes Polθ recruitment at sites of damage and alt-EJ repair. Finally, loss of FANCD2 in BRCA1/2-deficient tumors enhances cell death. These results reveal a synthetic lethal relationship between FANCD2 and BRCA1/2, and they identify FANCD2 as a central player orchestrating DNA repair pathway choice at the replication fork.

Differential Tus-Ter binding and lock formation: implications for DNA replication termination in Escherichia coli.

Science.gov (United States)

Moreau, Morgane J J; Schaeffer, Patrick M

2012-10-01

In E. coli, DNA replication termination occurs at Ter sites and is mediated by Tus. Two clusters of five Ter sites are located on each side of the terminus region and constrain replication forks in a polar manner. The polarity is due to the formation of the Tus-Ter-lock intermediate. Recently, it has been shown that DnaB helicase which unwinds DNA at the replication fork is preferentially stopped at the non-permissive face of a Tus-Ter complex without formation of the Tus-Ter-lock and that fork pausing efficiency is sequence dependent, raising two essential questions: Does the affinity of Tus for the different Ter sites correlate with fork pausing efficiency? Is formation of the Tus-Ter-lock the key factor in fork pausing? The combined use of surface plasmon resonance and GFP-Basta showed that Tus binds strongly to TerA-E and G, moderately to TerH-J and weakly to TerF. Out of these ten Ter sites only two, TerF and H, were not able to form significant Tus-Ter-locks. Finally, Tus's resistance to dissociation from Ter sites and the strength of the Tus-Ter-locks correlate with the differences in fork pausing efficiency observed for the different Ter sites by Duggin and Bell (2009).

Anaphase onset before complete DNA replication with intact checkpoint responses

DEFF Research Database (Denmark)

Torres-Rosell, Jordi; De Piccoli, Giacomo; Cordon-Preciado, Violeta

2007-01-01

Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most...

Replicating chromatin: a tale of histones

DEFF Research Database (Denmark)

Groth, Anja

2009-01-01

Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural...... framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...

Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts

Science.gov (United States)

Newman, Timothy J.; Mamun, Mohammed A.; Nieduszynski, Conrad A.; Blow, J. Julian

2013-01-01

During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts—Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe—also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10−8, which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms. PMID:23963700

Economic fork lifters

Energy Technology Data Exchange (ETDEWEB)

Roedig, W

1981-01-01

Increased energy costs attribute new interest to the choice between electric fork lifters and fork lifters driven by combustion engines. The advantages and shortcomings of the two drive systems are listed. As previous cost-comparisons are no longer up-to-date a new comparison is made in order to find out which of the three types works most economically: electric diesel- or fuel gas driven fork lifters. The comparison is based on a one-year-operation with a load capacity of 1,5 to 5 tons under normal stress conditions. The following parameters are compared: sum of investments, depreciation, interest rates, fixed costs per annum and per hour of operation. Variable costs like: repair costs, costs for replacement parts, energy cost and total cost. The three-wheeled electric fork lifter has proved to be the most economic one followed by the diesel-driven fork lifter, the four-wheel electric fork lifter and the fuel-gas driven fork lifter.

USP7/HAUSP: A SUMO deubiquitinase at the heart of DNA replication.

Science.gov (United States)

Smits, Veronique A J; Freire, Raimundo

2016-09-01

DNA replication is both highly conserved and controlled. Problematic DNA replication can lead to genomic instability and therefore carcinogenesis. Numerous mechanisms work together to achieve this tight control and increasing evidence suggests that post-translational modifications (phosphorylation, ubiquitination, SUMOylation) of DNA replication proteins play a pivotal role in this process. Here we discuss such modifications in the light of a recent article that describes a novel role for the deubiquitinase (DUB) USP7/HAUSP in the control of DNA replication. USP7 achieves this function by an unusual and novel mechanism, namely deubiquitination of SUMOylated proteins at the replication fork, making USP7 also a SUMO DUB (SDUB). This work extends previous observations of increased levels of SUMO and low levels of ubiquitin at the on-going replication fork. Here, we discuss this novel study, its contribution to the DNA replication and genomic stability field and what questions arise from this work. © 2016 WILEY Periodicals, Inc.

Replication dynamics of the yeast genome.

Science.gov (United States)

Raghuraman, M K; Winzeler, E A; Collingwood, D; Hunt, S; Wodicka, L; Conway, A; Lockhart, D J; Davis, R W; Brewer, B J; Fangman, W L

2001-10-05

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

DNA damage by X-rays and their impact on replication processes

International Nuclear Information System (INIS)

Parplys, Ann Christin; Petermann, Eva; Petersen, Cordula; Dikomey, Ekkehard; Borgmann, Kerstin

2012-01-01

Background: Replication-dependent radiosensitization of tumors ranks among the most promising tools for future improvements in tumor therapy. However, cell cycle checkpoint signaling during S phase is a key for maintaining genomic stability after ionizing irradiation allowing DNA damage repair by stabilizing replication forks, inhibiting new origin firing and recruiting DNA repair proteins. As the impact of the different types of DNA damage induced by ionizing radiation on replication fork functionality has not been investigated, this study was performed in tumor cells treated with various agents that induce specific DNA lesions. Methods: U2OS cells were exposed to methyl methanesulfonate (MMS) to induce base damage, low or high concentrations of hydrogen peroxide for the induction of SSBs, Topotecan to induce DSBs at replication, Mitomycin C (MMC) to induce interstrand cross-links or ionizing irradiation to analyze all damages. Chk1 phosphorylation, origin firing and replication fork progression, and cell cycle distribution were analyzed. Results: In our system, the extent of Chk1 phosphorylation was dependent on the type of damage induced and prolonged Chk1 phosphorylation correlated with the inhibition of replication initiation. Ionizing radiation, high concentrations of hydrogen peroxide, and Topotecan affected replication elongation much more strongly that the other agents. Almost all agents induced a slight increase in the S phase population but subsequent G2 arrest was only observed in response to those agents that strongly inhibited replication elongation and caused prolonged Chk1 phosphorylation. Conclusions: Our data suggest that to improve radiotherapy, radiosensitivity in S phase could be increased by combining irradiation with agents that induce secondary DSB or inhibit checkpoint signaling, such as inhibitors of PARP or Chk1.

Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

DEFF Research Database (Denmark)

Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

2014-01-01

The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies...... have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding...... yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells....

Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes.

Science.gov (United States)

Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

2014-01-01

The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells.

Two mechanisms coordinate replication termination by the Escherichia coli Tus–Ter complex

KAUST Repository

Pandey, Manjula; Elshenawy, Mohamed; Jergic, Slobodan; Takahashi, Masateru; Dixon, Nicholas E.; Hamdan, Samir; Patel, Smita S.

2015-01-01

The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7

Checkpoint independence of most DNA replication origins in fission yeast

OpenAIRE

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Abstract Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleo...

The Location of the Bacterial Origin of Replication is Critical for Initial Ciproflaxcin Antibiotic Resistance

Science.gov (United States)

Bos, Julia; Nehring, Ralph; Cruz, Diane; Austin, Doug; Rosenberg, Susan; Austin, Robert

By using E. coli cells in which the unique origin of replication has been moved to a ectopic chromosome location distant from the native one, we probe how perturbation of gene order near the origin of replication impacts genome stability and survival under genomic attack. We find that when challenged with sub-inhibitory doses of ciprofloxacin, an antibiotic that generates replication fork stalling, cells with the ectopic origin show significant fitness loss. We show that genes functionally relevant to the cipro-induced stress response are largely located near the native origin, even in distantly related species. We show that while cipro induces increased copy number of genes proximal to the origin of replication as a direct consequence of replication fork stalling, gene copy number variation was reduced near the ectopic origin. Altered gene dosage in cells with an ectopic origin resulted in impaired replication fork repair and chromosome instability. We propose that gene distribution in the origin region acts as a fundamental first line of defense when the integrity of the genome is threatened and that genes proximal to the origin of replication serve as a mechanism of genetic innovation and a driving force of genome evolution in the presence of genotoxic antibiotics. Lewis Sigler Institute for Integrative Genomics and the Physics Department at Princeton University.

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

Science.gov (United States)

Arbona, Jean-Michel; Goldar, Arach; Hyrien, Olivier; Arneodo, Alain; Audit, Benjamin

2018-06-01

The time-dependent rate I(t) of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the I(t) universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris . We predict the I(t) maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics. © 2018, Arbona et al.

Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination

DEFF Research Database (Denmark)

Vejrup-Hansen, Rasmus; Mizuno, Ken'Ichi; Miyabe, Izumi

2011-01-01

-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between ¿mms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that......-specific replication fork barrier and that, in a ¿mms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts...... is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site...

Mechanisms of bacterial DNA replication restart

Science.gov (United States)

Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

2018-01-01

Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

Science.gov (United States)

Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

2017-08-10

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

Science.gov (United States)

Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

2017-01-27

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

Science.gov (United States)

Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

2017-11-03

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

DEFF Research Database (Denmark)

Goldar, A.; Arneodo, A.; Audit, B.

2016-01-01

, and by taking into account the chromatin's fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement...

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

Science.gov (United States)

Goldar, Arach

2011-03-01

The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

Characterization of the Holliday junction resolving enzyme encoded by the Bacillus subtilis bacteriophage SPP1.

Directory of Open Access Journals (Sweden)

Lisa Zecchi

Full Text Available Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (oriL, and when replication progresses, the fork might stall by the binding of the origin binding protein G38P to the late replication origin (oriR. Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from P(E2 and P(E3 promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the P(E2 operon (genes 44 and 45 showed that they are in fact a single gene, re-annotated here as gene 44, that encodes a single polypeptide, named gene 44 product (G44P, 27.5 kDa. G44P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G44P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G44P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in B. subtilis cells. These in vitro and in vivo data suggest a role for G44P in replication restart during the transition to concatemeric viral replication.

The forked flap repair for hypospadias

Directory of Open Access Journals (Sweden)

Anil Chadha

2012-01-01

Full Text Available Context: Despite the abundance of techniques for the repair of Hypospadias, its problems still persist and a satisfactory design to correct the penile curvature with the formation of neourethra from the native urethral tissue or genital or extragenital tissues, with minimal postoperative complications has yet to evolve. Aim: Persisting with such an endeavor, a new technique for the repair of distal and midpenile hypospadias is described. Materials and Methods: The study has been done in 70 cases over the past 11 years. The "Forked-Flap" repair is a single stage method for the repair of such Hypospadias with chordee. It takes advantage of the rich vascular communication at the corona and capitalizes on the established reliability of the meatal based flip-flap. The repair achieves straightening of the curvature of the penis by complete excision of chordee tissue from the ventral surface of the penis beneath the urethral plate. The urethra is reconstructed using the native plate with forked flap extensions and genital tissue relying on the concept of meatal based flaps. Water proofing by dartos tissue and reinforcement by Nesbit′s prepucial tissue transfer completes the one stage procedure. Statistical Analysis: An analysis of 70 cases of this single stage technique of repair of penile hypospadias with chordee, operated at 3 to 5 years of age over the past 11 years is presented. Results and Conclusion: The Forked Flap gives comparable and replicable results; except for a urethrocutaneous fistula rate of 4% no other complications were observed.

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

NARCIS (Netherlands)

Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

2003-01-01

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA

p53 Maintains Genomic Stability by Preventing Interference between Transcription and Replication

Directory of Open Access Journals (Sweden)

Constance Qiao Xin Yeo

2016-04-01

Full Text Available p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression.

Genome-wide alterations of the DNA replication program during tumor progression

Science.gov (United States)

Arneodo, A.; Goldar, A.; Argoul, F.; Hyrien, O.; Audit, B.

2016-08-01

Oncogenic stress is a major driving force in the early stages of cancer development. Recent experimental findings reveal that, in precancerous lesions and cancers, activated oncogenes may induce stalling and dissociation of DNA replication forks resulting in DNA damage. Replication timing is emerging as an important epigenetic feature that recapitulates several genomic, epigenetic and functional specificities of even closely related cell types. There is increasing evidence that chromosome rearrangements, the hallmark of many cancer genomes, are intimately associated with the DNA replication program and that epigenetic replication timing changes often precede chromosomic rearrangements. The recent development of a novel methodology to map replication fork polarity using deep sequencing of Okazaki fragments has provided new and complementary genome-wide replication profiling data. We review the results of a wavelet-based multi-scale analysis of genomic and epigenetic data including replication profiles along human chromosomes. These results provide new insight into the spatio-temporal replication program and its dynamics during differentiation. Here our goal is to bring to cancer research, the experimental protocols and computational methodologies for replication program profiling, and also the modeling of the spatio-temporal replication program. To illustrate our purpose, we report very preliminary results obtained for the chronic myelogeneous leukemia, the archetype model of cancer. Finally, we discuss promising perspectives on using genome-wide DNA replication profiling as a novel efficient tool for cancer diagnosis, prognosis and personalized treatment.

RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication.

Science.gov (United States)

Vannier, Jean-Baptiste; Sandhu, Sumit; Petalcorin, Mark I R; Wu, Xiaoli; Nabi, Zinnatun; Ding, Hao; Boulton, Simon J

2013-10-11

Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles telomere loops (T loops) and suppresses telomere fragility to maintain the integrity of chromosome ends. We established that RTEL1 also associates with the replisome through binding to proliferating cell nuclear antigen (PCNA). Mouse cells disrupted for the RTEL1-PCNA interaction (PIP mutant) exhibited accelerated senescence, replication fork instability, reduced replication fork extension rates, and increased origin usage. Although T-loop disassembly at telomeres was unaffected in the mutant cells, telomere replication was compromised, leading to fragile sites at telomeres. RTEL1-PIP mutant mice were viable, but loss of the RTEL1-PCNA interaction accelerated the onset of tumorigenesis in p53-deficient mice. We propose that RTEL1 plays a critical role in both telomere and genome-wide replication, which is crucial for genetic stability and tumor avoidance.

RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

Science.gov (United States)

Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

2016-12-19

ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption

DEFF Research Database (Denmark)

Beck, Halfdan; Nähse-Kumpf, Viola; Larsen, Marie Sofie Yoo

2012-01-01

Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation of replic......Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation...... of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted...

Chromatin maturation depends on continued DNA-replication

International Nuclear Information System (INIS)

Schlaeger, E.J.; Puelm, W.; Knippers, R.

1983-01-01

The structure of [ 3 H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [ 3 H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. The authors conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation. (Auth.)

Broken silence restored-remodeling primes for deacetylation at replication forks

DEFF Research Database (Denmark)

Jasencakova, Zuzana; Groth, Anja

2011-01-01

Faithful propagation of chromatin structures requires assimilation of new histones to the modification profile of individual loci. In this issue of Molecular Cell, Rowbotham and colleagues identify a remodeler, SMARCAD1, acting at replication sites to facilitate histone deacetylation and restorat......Faithful propagation of chromatin structures requires assimilation of new histones to the modification profile of individual loci. In this issue of Molecular Cell, Rowbotham and colleagues identify a remodeler, SMARCAD1, acting at replication sites to facilitate histone deacetylation...

Replication stress, a source of epigenetic aberrations in cancer?

DEFF Research Database (Denmark)

Jasencakova, Zusana; Groth, Anja

2010-01-01

. Chromatin organization is transiently disrupted during DNA replication and maintenance of epigenetic information thus relies on faithful restoration of chromatin on the new daughter strands. Acute replication stress challenges proper chromatin restoration by deregulating histone H3 lysine 9 mono......-methylation on new histones and impairing parental histone recycling. This could facilitate stochastic epigenetic silencing by laying down repressive histone marks at sites of fork stalling. Deregulation of replication in response to oncogenes and other tumor-promoting insults is recognized as a significant source...... of genome instability in cancer. We propose that replication stress not only presents a threat to genome stability, but also jeopardizes chromatin integrity and increases epigenetic plasticity during tumorigenesis....

Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex.

Science.gov (United States)

Jaiswal, Rahul; Singh, Samarendra K; Bastia, Deepak; Escalante, Carlos R

2015-04-01

The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site. Batch crystallization under oil was required to produce crystals of good quality for data collection. Crystals grew in space group P2₁, with unit-cell parameters a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°. The crystals diffracted to a resolution of 3.0 Å. The crystals were mosaic and required two or three cycles of annealing. This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination.

A biologically inspired two-species exclusion model: effects of RNA polymerase motor traffic on simultaneous DNA replication

Science.gov (United States)

Ghosh, Soumendu; Mishra, Bhavya; Patra, Shubhadeep; Schadschneider, Andreas; Chowdhury, Debashish

2018-04-01

We introduce a two-species exclusion model to describe the key features of the conflict between the RNA polymerase (RNAP) motor traffic, engaged in the transcription of a segment of DNA, concomitant with the progress of two DNA replication forks on the same DNA segment. One of the species of particles (P) represents RNAP motors while the other (R) represents the replication forks. Motivated by the biological phenomena that this model is intended to capture, a maximum of two R particles only are allowed to enter the lattice from two opposite ends whereas the unrestricted number of P particles constitutes a totally asymmetric simple exclusion process (TASEP) in a segment in the middle of the lattice. The model captures three distinct pathways for resolving the co-directional as well as head-on collision between the P and R particles. Using Monte Carlo simulations and heuristic analytical arguments that combine exact results for the TASEP with mean-field approximations, we predict the possible outcomes of the conflict between the traffic of RNAP motors (P particles engaged in transcription) and the replication forks (R particles). In principle, the model can be adapted to experimental conditions to account for the data quantitatively.

Mms1 binds to G-rich regions in Saccharomyces cerevisiae and influences replication and genome stability

NARCIS (Netherlands)

Wanzek, Katharina; Schwindt, Eike; Capra, John A.; Paeschke, Katrin

2017-01-01

The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and

Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

DEFF Research Database (Denmark)

Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

2007-01-01

Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chro......Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division......, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple......-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive...

G-Quadruplexes in DNA Replication: A Problem or a Necessity?

Science.gov (United States)

Valton, Anne-Laure; Prioleau, Marie-Noëlle

2016-11-01

DNA replication is a highly regulated process that ensures the correct duplication of the genome at each cell cycle. A precise cell type-specific temporal program controls the duplication of complex vertebrate genomes in an orderly manner. This program is based on the regulation of both replication origin firing and replication fork progression. G-quadruplexes (G4s), DNA secondary structures displaying noncanonical Watson-Crick base pairing, have recently emerged as key controllers of genome duplication. Here we discuss the various means by which G4s affect this fundamental cellular process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mycobacterium tuberculosis RecG binds and unwinds model DNA substrates with a preference for Holliday junctions.

Science.gov (United States)

Zegeye, Ephrem Debebe; Balasingham, Seetha V; Laerdahl, Jon K; Homberset, Håvard; Tønjum, Tone

2012-08-01

The RecG enzyme, a superfamily 2 helicase, is present in nearly all bacteria. Here we report for the first time that the recG gene is also present in the genomes of most vascular plants as well as in green algae, but is not found in other eukaryotes or archaea. The precise function of RecG is poorly understood, although ample evidence shows that it plays critical roles in DNA repair, recombination and replication. We further demonstrate that Mycobacterium tuberculosis RecG (RecG(Mtb)) DNA binding activity had a broad substrate specificity, whereas it only unwound branched-DNA substrates such as Holliday junctions (HJs), replication forks, D-loops and R-loops, with a strong preference for the HJ as a helicase substrate. In addition, RecG(Mtb) preferentially bound relatively long (≥40 nt) ssDNA, exhibiting a higher affinity for the homopolymeric nucleotides poly(dT), poly(dG) and poly(dC) than for poly(dA). RecG(Mtb) helicase activity was supported by hydrolysis of ATP or dATP in the presence of Mg(2+), Mn(2+), Cu(2+) or Fe(2+). Like its Escherichia coli orthologue, RecG(Mtb) is also a strictly DNA-dependent ATPase.

DNA replication: stalling a fork for imprinting and switching

DEFF Research Database (Denmark)

Egel, Richard

2004-01-01

Mating-type switching in fission yeast has long been known to be directed by a DNA 'imprint'. This imprint has now been firmly characterized as a protected site-specific and strand-specific nick. New work also links the widely conserved Swi1-Swi3 complex to the protection of stalled replication...

Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy.

Science.gov (United States)

Zellweger, Ralph; Lopes, Massimo

2018-01-01

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.

Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

DEFF Research Database (Denmark)

Munk, Stephanie; Sigurðsson, Jón Otti; Xiao, Zhenyu

2017-01-01

The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essentia....... They analyze changes in the SUMO and phosphoproteome after MMC and hydroxyurea treatments and find that the DNA damage response kinases ATR and ATM globally regulate SUMOylation upon replication stress and fork breakage....

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

Science.gov (United States)

Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

2015-12-01

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

South Fork Holston River basin 1988 biomonitoring

Energy Technology Data Exchange (ETDEWEB)

Saylor, C.F.; Ahlstedt, S.A.

1990-06-01

There is concern over the effects of shifts in land use use practices on the aquatic fauna of streams in the South Fork Holston River basin in northwestern North Carolina and southwestern Virginia. Trout reproduction has noticeably declined in the Watauga River subbasin. The Watauga River and Elk River subbasins have been subjected to commercial and resort development. The Middle fork Holston River and the upper South Fork Holston River subbasins have been affected by agricultural and mining activities, respectively (Cox, 1986). To aid reclamation and management of the South Fork Holston basin, Tennessee Valley Authority (TVA) biologists conducted biomonitoring--including index of biotic integrity and macroinvertebrate sampling--on the Middle Fork Holston, South Fork Holston, Watauga, and Elk Rivers to assess cumulative impairment related to changes in habitat and pollutant loading in these subbasins. Biomonitoring can detect environmental degradation, help document problem areas, and assist in development of strategies for managing water quality. This report discusses the methods and materials and results of the biomonitoring of South Fork Holston River Basin. 13 refs., 5 figs., 12 tabs.

RADX interacts with single-stranded DNA to promote replication fork stability

DEFF Research Database (Denmark)

Schubert, Lisa; Ho, Teresa; Hoffmann, Saskia

2017-01-01

Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex...... ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins....

Checkpoint independence of most DNA replication origins in fission yeast.

Science.gov (United States)

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-12-19

In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (approximately 3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint

Checkpoint independence of most DNA replication origins in fission yeast

Science.gov (United States)

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in

Checkpoint independence of most DNA replication origins in fission yeast

Directory of Open Access Journals (Sweden)

Scott Donna

2007-12-01

Full Text Available Abstract Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU, which limits the pool of deoxyribonucleoside triphosphates (dNTPs and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR or cds1 (which encodes the fission yeast homologue of Chk2. Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3% behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

Directory of Open Access Journals (Sweden)

Simon Gemble

2015-07-01

Full Text Available Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS, a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

Science.gov (United States)

Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-12-01

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Code Forking, Governance, and Sustainability in Open Source Software

Directory of Open Access Journals (Sweden)

Juho Lindman

2013-01-01

Full Text Available The right to fork open source code is at the core of open source licensing. All open source licenses grant the right to fork their code, that is to start a new development effort using an existing code as its base. Thus, code forking represents the single greatest tool available for guaranteeing sustainability in open source software. In addition to bolstering program sustainability, code forking directly affects the governance of open source initiatives. Forking, and even the mere possibility of forking code, affects the governance and sustainability of open source initiatives on three distinct levels: software, community, and ecosystem. On the software level, the right to fork makes planned obsolescence, versioning, vendor lock-in, end-of-support issues, and similar initiatives all but impossible to implement. On the community level, forking impacts both sustainability and governance through the power it grants the community to safeguard against unfavourable actions by corporations or project leaders. On the business-ecosystem level forking can serve as a catalyst for innovation while simultaneously promoting better quality software through natural selection. Thus, forking helps keep open source initiatives relevant and presents opportunities for the development and commercialization of current and abandoned programs.

Conflict Resolution in the Genome: How Transcription and Replication Make It Work.

Science.gov (United States)

Hamperl, Stephan; Cimprich, Karlene A

2016-12-01

The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

Science.gov (United States)

Tian, Yanyan; Shen, Xi; Wang, Rui; Klages-Mundt, Naeh L; Lynn, Erica J; Martin, Sara K; Ye, Yin; Gao, Min; Chen, Junjie; Schlacher, Katharina; Li, Lei

2017-12-08

In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Code Forking, Governance, and Sustainability in Open Source Software

OpenAIRE

Juho Lindman; Linus Nyman

2013-01-01

The right to fork open source code is at the core of open source licensing. All open source licenses grant the right to fork their code, that is to start a new development effort using an existing code as its base. Thus, code forking represents the single greatest tool available for guaranteeing sustainability in open source software. In addition to bolstering program sustainability, code forking directly affects the governance of open source initiatives. Forking, and even the mere possibilit...

Timing, Coordination, and Rhythm : Acrobatics at the DNA Replication Fork

NARCIS (Netherlands)

Hamdan, Samir M.; Oijen, Antoine M. van

2010-01-01

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In

Initiation preference at a yeast origin of replication.

Science.gov (United States)

Brewer, B J; Fangman, W L

1994-04-12

Replication origins in the yeast Saccharomyces cerevisiae are identified as autonomous replication sequence (ARS) elements. To examine the effect of origin density on replication initiation, we have analyzed the replication of a plasmid that contains two copies of the same origin, ARS1. The activation of origins and the direction that replication forks move through flanking sequences can be physically determined by analyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacing of ARSs results in an apparent interference between the potential origins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently than the other one. These results show that the plasmid context is critical for determining the preferred origin. This origin preference is also exhibited when the tandem copies of ARS1 are introduced into a yeast chromosome. The sequences responsible for establishing the origin preference have been identified by deletion analysis and are found to reside in a portion of the yeast URA3 gene.

Gene organization inside replication domains in mammalian genomes

Science.gov (United States)

Zaghloul, Lamia; Baker, Antoine; Audit, Benjamin; Arneodo, Alain

2012-11-01

We investigate the large-scale organization of human genes with respect to "master" replication origins that were previously identified as bordering nucleotide compositional skew domains. We separate genes in two categories depending on their CpG enrichment at the promoter which can be considered as a marker of germline DNA methylation. Using expression data in mouse, we confirm that CpG-rich genes are highly expressed in germline whereas CpG-poor genes are in a silent state. We further show that, whether tissue-specific or broadly expressed (housekeeping genes), the CpG-rich genes are over-represented close to the replication skew domain borders suggesting some coordination of replication and transcription. We also reveal that the transcription of the longest CpG-rich genes is co-oriented with replication fork progression so that the promoter of these transcriptionally active genes be located into the accessible open chromatin environment surrounding the master replication origins that border the replication skew domains. The observation of a similar gene organization in the mouse genome confirms the interplay of replication, transcription and chromatin structure as the cornerstone of mammalian genome architecture.

USP7 is a SUMO deubiquitinase essential for DNA replication

DEFF Research Database (Denmark)

Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia

2016-01-01

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment...... is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads...... to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7...

SC tuning fork

CERN Document Server

The tuning fork used to modulate the radiofrequency system of the synchro cyclotron (SC) from 1957 to 1973. This piece is an unused spare part. The SC was the 1st accelerator built at CERN. It operated from August 1957 until it was closed down at the end of 1990. In the SC the magnetic field did not change with time, and the particles were accelerated in successive pulses by a radiofrequency voltage of some 20kV which varied in frequency as they spiraled outwards towards the extraction radius. The frequency varied from 30MHz to about 17Mz in each pulse. The tuning fork vibrated at 55MHz in vacuum in an enclosure which formed a variable capacitor in the tuning circuit of the RF system, allowing the RF to vary over the appropriate range to accelerate protons from the centre of the macine up to 600Mev at extraction radius. In operation the tips of the tuning fork blade had an amplitude of movement of over 1 cm. The SC accelerator underwent extensive improvements from 1973 to 1975, including the installation of a...

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication.

Directory of Open Access Journals (Sweden)

Dany Graindorge

Full Text Available UVA radiation (320-400 nm is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS, such as singlet oxygen (1O2 and hydrogen peroxide (H2O2, which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1 to several hours (replication fork velocity and origin firing. The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication

Science.gov (United States)

Graindorge, Dany; Martineau, Sylvain; Machon, Christelle; Arnoux, Philippe; Guitton, Jérôme; Francesconi, Stefania; Frochot, Céline; Sage, Evelyne; Girard, Pierre-Marie

2015-01-01

UVA radiation (320–400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydrogen peroxide (H2O2), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen. PMID:26485711

Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing*S⃞

Science.gov (United States)

Thiriet, Christophe; Hayes, Jeffrey J.

2009-01-01

Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing. PMID:19015270

USP7 is a SUMO deubiquitinase essential for DNA replication

Science.gov (United States)

Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia; Lopez-Contreras, Andres J; Ruppen, Isabel; Murga, Matilde; Muñoz, Javier; Mendez, Juan; Fernandez-Capetillo, Oscar

2016-01-01

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates various aspects of DNA replication. We previously showed that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/Ub-low environment is maintained at sites of DNA replication is not known. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced to chromatin away from replisomes. Our findings provide a model to explain the differential accumulation of SUMO and Ub at replication forks, and identify an essential role of USP7 in DNA replication that should be taken into account for the use of USP7 inhibitors as anticancer agents. PMID:26950370

Asynchronous DNA replication within the human β-globin gene locus

International Nuclear Information System (INIS)

Epner, E.; Forrester, W.C.; Groudine, M.

1988-01-01

The timing of DNA replication of the human β-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human β-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-γ-globin gene region and approximately 20 kilobases 5' to the ε-globin gene and 20 kilobases 3' to the β-globin gene, replicate later and throughout S phase. A similar area is also present in the α-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks

Mcm2 phosphorylation and the response to replicative stress

Directory of Open Access Journals (Sweden)

Stead Brent E

2012-05-01

Full Text Available Abstract Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm proteins 2 through 7 (Mcm2-7 and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK. In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU and to the base analogue 5-fluorouracil (5-FU but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

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Goldar, A.; Arneodo, A.; Audit, B.; Argoul, F.; Rappailles, A.; Guilbaud, G.; Petryk, N.; Kahli, M.; Hyrien, O.

2016-03-01

We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin’s fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

Forked and Integrated Variants In An Open-Source Firmware Project

DEFF Research Database (Denmark)

Stanciulescu, Stefan; Schulze, Sandro; Wasowski, Andrzej

2015-01-01

and interactive source management platforms such as Github. We study advantages and disadvantages of forking using the case of Marlin, an open source firmware for 3D printers. We find that many problems and advantages of cloning do translate to forking. Interestingly, the Marlin community uses both forking......Code cloning has been reported both on small (code fragments) and large (entire projects) scale. Cloning-in-the-large, or forking, is gaining ground as a reuse mechanism thanks to availability of better tools for maintaining forked project variants, hereunder distributed version control systems...

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

Science.gov (United States)

Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

2016-04-07

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

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

Science.gov (United States)

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

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

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

Science.gov (United States)

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

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

Replicative Stress Induces Intragenic Transcription of the ASE1 Gene that Negatively Regulates Ase1 Activity

OpenAIRE

McKnight, Kelly; Liu, Hong; Wang, Yanchang

2014-01-01

Intragenic transcripts initiate within the coding region of a gene, thereby producing shorter mRNAs and proteins. Although intragenic transcripts are widely expressed [1], their role in the functional regulation of genes remains largely unknown. In budding yeast, DNA replication stress activates the S-phase checkpoint that stabilizes replication forks and arrests cells in S-phase with a short spindle [2-4]. When yeast cells were treated with hydroxyurea (HU) to block DNA synthesis and induce ...

Inhibition of DNA replication by ultraviolet light

International Nuclear Information System (INIS)

Edenberg, H.J.

1976-01-01

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10 J/m 2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers

Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

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Thakur, Roshan S; Basavaraju, Shivakumar; Somyajit, Kumar; Jain, Akshatha; Subramanya, Shreelakshmi; Muniyappa, Kalappa; Nagaraju, Ganesh

2013-04-01

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions. © 2013 The Authors Journal compilation © 2013 FEBS.

RAD51 interconnects between DNA replication, DNA repair and immunity.

Science.gov (United States)

Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

2017-05-05

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of Tuning Fork Based Probes for Atomic Force Microscopy

Science.gov (United States)

Jalilian, Romaneh; Yazdanpanah, Mehdi M.; Torrez, Neil; Alizadeh, Amirali; Askari, Davood

2014-03-01

This article reports on the development of tuning fork-based AFM/STM probes in NaugaNeedles LLC for use in atomic force microscopy. These probes can be mounted on different carriers per customers' request. (e.g., RHK carrier, Omicron carrier, and tuning fork on a Sapphire disk). We are able to design and engineer tuning forks on any type of carrier used in the market. We can attach three types of tips on the edge of a tuning fork prong (i.e., growing Ag2Ga nanoneedles at any arbitrary angle, cantilever of AFM tip, and tungsten wire) with lengths from 100-500 μm. The nanoneedle is located vertical to the fork. Using a suitable insulation and metallic coating, we can make QPlus sensors that can detect tunneling current during the AFM scan. To make Qplus sensors, the entire quartz fork will be coated with an insulating material, before attaching the nanoneedle. Then, the top edge of one prong is coated with a thin layer of conductive metal and the nanoneedle is attached to the fork end of the metal coated prong. The metal coating provides electrical connection to the tip for tunneling current readout and to the electrodes and used to read the QPlus current. Since the amount of mass added to the fork is minimal, the resonance frequency spectrum does not change and still remains around 32.6 KHz and the Q factor is around 1,200 in ambient condition. These probes can enhance the performance of tuning fork based atomic microscopy.

Burnup verification using the FORK measurement system

International Nuclear Information System (INIS)

Ewing, R.I.

1994-01-01

Verification measurements may be used to help ensure nuclear criticality safety when burnup credit is applied to spent fuel transport and storage systems. The FORK measurement system, designed at Los Alamos National Laboratory for the International Atomic Energy Agency safeguards program, has been used to verify reactor site records for burnup and cooling time for many years. The FORK system measures the passive neutron and gamma-ray emission from spent fuel assemblies while in the storage pool. This report deals with the application of the FORK system to burnup credit operations based on measurements performed on spent fuel assemblies at the Oconee Nuclear Station of Duke Power Company

Take It Slow: can feedback from a smart fork reduce eating speed?

Directory of Open Access Journals (Sweden)

Sander Hermsen

2015-09-01

The present study examines the efficacy of a smart fork that helps people to eat more slowly. This adapted fork records eating speed and delivers vibrotactile feedback if users eat too quickly. In two studies, we tested the acceptability and user experience of the fork (Study 1, and its effect on eating rate and satiety levels in a controlled lab-setting (Study 2. Method: In study 1, 11 participants (all self-reported fast eaters ate a meal using the fork in our laboratory and used the fork for three consecutive days in their home setting. Participants took part in semi-structured interviews after the first meal and upon returning the fork, covering perceived effect on eating rate, comfort of use, accuracy, and motivational and social aspects of fork use. Interviews were coded and a thematic classification analysis was performed. In study 2, 128 participants (all self-reported fast eaters ate a standardized meal using the fork in our laboratory. We used a between-participants design with 2 conditions; participants ate their meal either with vibrotactile feedback from the fork (experimental condition or ate their meal without vibrotactile feedback (control condition. Eating rate, meal duration, error rate (number of bites taken faster than 10 seconds after previous bite, total food intake, and satiety were recorded for every participant. Results: Study 1: All participants felt that the feedback was generally accurate and consistent. Fork size, weight, and intensity of the feedback were seen as comfortable and acceptable. All participants reported a heightened awareness of eating rate and all but one participant reported eating more slowly with the fork. Study 2: Participants in the experimental condition ate significantly slower, and with a lower error rate than those in the control condition. Feedback did not significantly affect the amount of food eaten. Conclusions: Our findings suggest that this smart fork is an acceptable and effective tool to disrupt and

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

Science.gov (United States)

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

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

Science.gov (United States)

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

2014-01-01

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

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

Directory of Open Access Journals (Sweden)

Gregory A Sowd

2014-12-01

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

The kinase domain residue serine 173 of Schizosaccharomyces pombe Chk1 kinase is critical for the response to DNA replication stress

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Naomi Coulton

2017-12-01

Full Text Available While mammalian Chk1 kinase regulates replication origins, safeguards fork integrity and promotes fork progression, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A in the kinase domain of fission yeast Chk1 abolishes the G1-M and S-M checkpoints with little impact on the G2-M arrest. This separation-of-function mutation strongly reduces the Rad3-dependent phosphorylation of Chk1 at serine 345 during logarithmic growth, but not when cells experience exogenous DNA damage. Loss of S173 lowers the restrictive temperature of a catalytic DNA polymerase epsilon mutant (cdc20.M10 and is epistatic with a mutation in DNA polymerase delta (cdc6.23 when DNA is alkylated by methyl-methanesulfate (MMS. The chk1-S173A allele is uniquely sensitive to high MMS concentrations where it displays a partial checkpoint defect. A complete checkpoint defect occurs only when DNA replication forks break in cells without the intra-S phase checkpoint kinase Cds1. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 (cdc10.V50 is impaired. We conclude that serine 173, which is equivalent to lysine 166 in the activation loop of human Chk1, is only critical in DNA polymerase mutants or when forks collapse in the absence of Cds1.

Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

Science.gov (United States)

Kunkel, Thomas A; Burgers, Peter M J

2017-08-01

Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

0615-0641_ESM.doc

Indian Academy of Sciences (India)

ODN7 and ODN17. 80-bp DNA. ODN8 and ODN18. 90-bp DNA. ODN9 and ODN19. 100-bp DNA. ODN10 and ODN20. Holliday junction. ODN21, ODN 22, ODN23 and ODN24. Replication fork. ODN 22, ODN 23, ODN 5 and ODN 6. 3' Flap. ODN 22, ODN 23 and ODN26. 5' Flap. ODN 22, ODN 23 and ODN27. 5' Overhang.

RECQ5 helicase promotes resolution of conflicts between replication and transcription in human cells

Czech Academy of Sciences Publication Activity Database

Urban, Václav; Dobrovolná, Jana; Hühn, D.; Fryzelkova, Jana; Bartek, Jiří; Janščák, Pavel

2016-01-01

Roč. 214, č. 4 (2016), s. 401-415 ISSN 0021-9525 R&D Projects: GA ČR(CZ) GA14-05743S; GA MŠk LH14037 Institutional support: RVO:68378050 Keywords : rna-polymerase-ii * fragile sites * homologous recombination * genome instability * dna-replication * genes * fork * protein * stress * brca1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.955, year: 2016

Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

DEFF Research Database (Denmark)

Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Po

2014-01-01

To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics during...... replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors...... such as CAF-1, DNMT1 and SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment...

Global profiling of DNA replication timing and efficiency reveals that efficient replication/firing occurs late during S-phase in S. pombe.

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Majid Eshaghi

Full Text Available BACKGROUND: During S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Delta cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Delta cells after HU removal, owing to the firing at the remaining unused (inefficient origins during HU treatment. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.

Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor.

Directory of Open Access Journals (Sweden)

Andrei Kuzminov

2016-10-01

Full Text Available As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i increased chromosomal fragmentation and (ii complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF. To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication. In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.

FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress

DEFF Research Database (Denmark)

Fugger, Kasper; Chu, Wai Kit; Haahr, Peter

2013-01-01

The molecular events occurring following the disruption of DNA replication forks are poorly characterized, despite extensive use of replication inhibitors such as hydroxyurea in the treatment of malignancies. Here, we identify a key role for the FBH1 helicase in mediating DNA double-strand break...... formation following replication inhibition. We show that FBH1-deficient cells are resistant to killing by hydroxyurea, and exhibit impaired activation of the pro-apoptotic factor p53, consistent with decreased DNA double-strand break formation. Similar findings were obtained in murine ES cells carrying...... of replication stress. Our data suggest that FBH1 helicase activity is required to eliminate cells with excessive replication stress through the generation of MUS81-induced DNA double-strand breaks....

Chronic DNA Replication Stress Reduces Replicative Lifespan of Cells by TRP53-Dependent, microRNA-Assisted MCM2-7 Downregulation.

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Gongshi Bai

2016-01-01

Full Text Available Circumstances that compromise efficient DNA replication, such as disruptions to replication fork progression, cause a state known as DNA replication stress (RS. Whereas normally proliferating cells experience low levels of RS, excessive RS from intrinsic or extrinsic sources can trigger cell cycle arrest and senescence. Here, we report that a key driver of RS-induced senescence is active downregulation of the Minichromosome Maintenance 2-7 (MCM2-7 factors that are essential for replication origin licensing and which constitute the replicative helicase core. Proliferating cells produce high levels of MCM2-7 that enable formation of dormant origins that can be activated in response to acute, experimentally-induced RS. However, little is known about how physiological RS levels impact MCM2-7 regulation. We found that chronic exposure of primary mouse embryonic fibroblasts (MEFs to either genetically-encoded or environmentally-induced RS triggered gradual MCM2-7 repression, followed by inhibition of replication and senescence that could be accelerated by MCM hemizygosity. The MCM2-7 reduction in response to RS is TRP53-dependent, and involves a group of Trp53-dependent miRNAs, including the miR-34 family, that repress MCM expression in replication-stressed cells before they undergo terminal cell cycle arrest. miR-34 ablation partially rescued MCM2-7 downregulation and genomic instability in mice with endogenous RS. Together, these data demonstrate that active MCM2-7 repression is a physiologically important mechanism for RS-induced cell cycle arrest and genome maintenance on an organismal level.

Replication stress interferes with histone recycling and predeposition marking of new histones

DEFF Research Database (Denmark)

Jasencakova, Zuzana; Scharf, Annette N D; Ask, Katrine

2010-01-01

To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified...... remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest......, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows...

Improve performance of scanning probe microscopy by balancing tuning fork prongs

International Nuclear Information System (INIS)

Ng, Boon Ping; Zhang Ying; Wei Kok, Shaw; Chai Soh, Yeng

2009-01-01

This paper presents an approach for improving the Q-factor of tuning fork probe used in scanning probe microscopes. The improvement is achieved by balancing the fork prongs with extra mass attachment. An analytical model is proposed to characterize the Q-factor of a tuning fork probe with respect to the attachment of extra mass on the tuning fork prongs, and based on the model, the Q-factors of the unbalanced and balanced tuning fork probes are derived and compared. Experimental results showed that the model fits well the experimental data and the approach can improve the Q-factor by more than a factor of three. The effectiveness of the approach is further demonstrated by applying the balanced probe on an atomic force microscope to obtain improved topographic images.

DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks

DEFF Research Database (Denmark)

Mosbech, Anna; Gibbs-Seymour, Ian; Kagias, Konstantinos

2012-01-01

Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress...... synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage...

The participation of the Fanconi anemia pathway in the replication of UV-damage DNA

International Nuclear Information System (INIS)

Federico, M.B.; Vallerga, M.B.; Mansilla, S.F.; Speroni, J.; Habif, M.; D'Alessio, C.; Gottifredi, V.

2011-01-01

When cells are challenged with genotoxic agents, replicating cells must use damaged DNA as templates. In this way, active replication forks do not collapse and cell viability is protected. After UV irradiation a specialized DNA polymerase pol eta uses UV damaged DNA as template. Intriguingly, Pol eta lost in human cells does not steeply increase UV sensitivity. This suggests that compensatory mechanisms promote cell survival when pol eta is absent. We have found an increase and sustained FANCD2 ubiquitination and focal formation after UV irradiation when pol eta is lost. FANCD2 is a key marker of the activation of the FANCONI ANEMIA (FA) pathway. While there is limited information regarding a role of the FA pathway after UV irradiation, it is well established that FANCD2 ubiquitination is linked to the recruitment of homologous recombination (HR) specific markers to other lesions. We therefore thought that cell viability in the absence of pol eta might result from the activation of FANDC2-dependent HR at collapsed replication forks. We are currently analyzing markers of damage such as γH2AX phosphorylation, markers of HR such as Rad51, markers of double strand breaks accumulation such as 53BP1 and setting up viability assays. This information might allow us to predict if FANCD2 can trigger HR after UV and if this contributes to cell viability when pol eta is absent. (authors)

Timeless links replication termination to mitotic kinase activation.

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Jayaraju Dheekollu

2011-05-01

Full Text Available The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1. Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

The fecundity of fork-tailed threadfin bream (Nemipterus furcosus) in Bangka, Bangka Belitung

Science.gov (United States)

Utami, E.; Safitriyani, E.; Gatra Persada, Leo

2018-04-01

Fork-tailed threadfin bream (Nemipterus furcosus) is one of important economic fishes in Bangka. The sustainability of fork-tailed threadfin bream is threatened by degradation of natural habitat. Information of reproductive is needed for further management. The objective of this study was to examine fecundity of fork-tailed threadfin bream. The mean values of temperature was 28.83 ± 0,37°C, respectively. Sex ratio during sampling showed that female fork-tailed threadfin bream greater than male population. Berried female fork-tailed threadfin bream found from March until November. The greatest number of berried female fork-tailed threadfin bream showed in July with berried female value of 25. Fork-tailed threadfin bream fecundity was 19951 and 66628, respectively. The fecundity data can be used to access the reproductive potential of fish stock and also as an assessment on stock size of their natural population.

Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

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Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

2014-10-01

To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

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Stephanie Munk

2017-10-01

Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

DNA replication stress restricts ribosomal DNA copy number.

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Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

DNA replication stress restricts ribosomal DNA copy number

Science.gov (United States)

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

DNA replication stress restricts ribosomal DNA copy number.

Directory of Open Access Journals (Sweden)

Devika Salim

2017-09-01

Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

Science.gov (United States)

Acevedo, Julyana; Yan, Shan; Michael, W. Matthew

2016-01-01

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

Direct non transcriptional role of NF-Y in DNA replication.

Science.gov (United States)

Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

2016-04-01

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

Science.gov (United States)

Macheret, Morgane; Halazonetis, Thanos D

2018-03-01

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

Science.gov (United States)

Huang, Hongda; Strømme, Caroline B; Saredi, Giulia; Hödl, Martina; Strandsby, Anne; González-Aguilera, Cristina; Chen, Shoudeng; Groth, Anja; Patel, Dinshaw J

2015-08-01

During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

South Fork Snake River/Palisades Wildlife Mitigation Project: Environmental assessment

Energy Technology Data Exchange (ETDEWEB)

NONE

1995-09-01

BPA proposes to fund the implementation of the South Fork Snake River Programmatic Management Plan to compensate for losses of wildlife and wildlife habitat due to hydroelectric development at Palisades Dam. The Idaho Department of Fish and Game drafted the plan, which was completed in May 1993. This plan recommends land and conservation easement acquisition and wildlife habitat enhancement measures. These measures would be implemented on selected lands along the South Fork of the Snake River between Palisades Dam and the confluence with the Henry`s Fork, and on portions of the Henry`s Fork located in Bonneville, Madison, and Jefferson Counties, Idaho. BPA has prepared an Environmental Assessment evaluating the proposed project. The EA also incorporates by reference the analyses in the South Fork Snake River Activity/Operations Plan and EA prepared jointly in 1991 by the Bureau of Land Management and the Forest Service. Based on the analysis in the EA, BPA has determined that the proposed action is not a major Federal action significantly affecting the quality of the human environment within the meaning of the National Environmental Policy Act (NEPA) of 1969. Therefore, the preparation of an Environmental Impact Statement (EIS) is not required and BPA is issuing this FONSI.

Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation

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Takashi Kubota

2015-08-01

Full Text Available The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.

Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution.

Science.gov (United States)

Gahlon, Hailey L; Romano, Louis J; Rueda, David

2017-11-20

Faithful replication of DNA is a critical aspect in maintaining genome integrity. DNA polymerases are responsible for replicating DNA, and high-fidelity polymerases do this rapidly and at low error rates. Upon exposure to exogenous or endogenous substances, DNA can become damaged and this can alter the speed and fidelity of a DNA polymerase. In this instance, DNA polymerases are confronted with an obstacle that can result in genomic instability during replication, for example, by nucleotide misinsertion or replication fork collapse. It is important to know how DNA polymerases respond to damaged DNA substrates to understand the mechanism of mutagenesis and chemical carcinogenesis. Single-molecule techniques have helped to improve our current understanding of DNA polymerase-mediated DNA replication, as they enable the dissection of mechanistic details that can otherwise be lost in ensemble-averaged experiments. These techniques have also been used to gain a deeper understanding of how single DNA polymerases behave at the site of the damage in a DNA substrate. In this review, we evaluate single-molecule studies that have examined the interaction between DNA polymerases and damaged sites on a DNA template.

Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

International Nuclear Information System (INIS)

Liu, J.-S.; Kuo, S.-R.; Melendy, Thomas

2003-01-01

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of γ-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of γ-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced γ-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression

Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

Energy Technology Data Exchange (ETDEWEB)

Liu, J.-S.; Kuo, S.-R.; Melendy, Thomas

2003-11-27

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of {gamma}-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of {gamma}-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced {gamma}-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.

Advancing the Fork detector for quantitative spent nuclear fuel verification

Science.gov (United States)

Vaccaro, S.; Gauld, I. C.; Hu, J.; De Baere, P.; Peterson, J.; Schwalbach, P.; Smejkal, A.; Tomanin, A.; Sjöland, A.; Tobin, S.; Wiarda, D.

2018-04-01

The Fork detector is widely used by the safeguards inspectorate of the European Atomic Energy Community (EURATOM) and the International Atomic Energy Agency (IAEA) to verify spent nuclear fuel. Fork measurements are routinely performed for safeguards prior to dry storage cask loading. Additionally, spent fuel verification will be required at the facilities where encapsulation is performed for acceptance in the final repositories planned in Sweden and Finland. The use of the Fork detector as a quantitative instrument has not been prevalent due to the complexity of correlating the measured neutron and gamma ray signals with fuel inventories and operator declarations. A spent fuel data analysis module based on the ORIGEN burnup code was recently implemented to provide automated real-time analysis of Fork detector data. This module allows quantitative predictions of expected neutron count rates and gamma units as measured by the Fork detectors using safeguards declarations and available reactor operating data. This paper describes field testing of the Fork data analysis module using data acquired from 339 assemblies measured during routine dry cask loading inspection campaigns in Europe. Assemblies include both uranium oxide and mixed-oxide fuel assemblies. More recent measurements of 50 spent fuel assemblies at the Swedish Central Interim Storage Facility for Spent Nuclear Fuel are also analyzed. An evaluation of uncertainties in the Fork measurement data is performed to quantify the ability of the data analysis module to verify operator declarations and to develop quantitative go/no-go criteria for safeguards verification measurements during cask loading or encapsulation operations. The goal of this approach is to provide safeguards inspectors with reliable real-time data analysis tools to rapidly identify discrepancies in operator declarations and to detect potential partial defects in spent fuel assemblies with improved reliability and minimal false positive alarms

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

Science.gov (United States)

Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

2011-01-01

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

Channelization and floodplain forests: impacts of accelerated sedimentation and valley plug formation on floodplain forests of the Middle Fork Forked Deer River, Tennessee, USA

Science.gov (United States)

Sonja N. Oswalt; Sammy L. King

2005-01-01

We evaluated the severe degradation of floodplain habitats resulting from channelization and concomitant excessive coarse sedimentation on the Middle Fork Forked Deer River in west Tennessee from 2000 to 2003. Land use practices have resulted in excessive sediment in the tributaries and river system eventually resulting in sand deposition on the floodplain, increased...

Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

Science.gov (United States)

Chastain, Megan; Zhou, Qing; Shiva, Olga; Fadri-Moskwik, Maria; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Ye, Ping; Chai, Weihang

2016-08-02

The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

DNA fork displacement rates in human cells

International Nuclear Information System (INIS)

Kapp, L.N.; Painter, R.B.

1981-01-01

DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 μm/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions. (Auth.)

DNA fork displacement rates in human cells

Energy Technology Data Exchange (ETDEWEB)

Kapp, L.N.; Painter, R.B. (California Univ., San Francisco (USA). Lab. of Radiobiology)

1981-11-27

DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 ..mu..m/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions.

RecQ helicases and cellular responses to DNA damage

International Nuclear Information System (INIS)

Wu, Leonard; Hickson, Ian D.

2002-01-01

The faithful replication of the genome is essential for the survival of all organisms. It is not surprising therefore that numerous mechanisms have evolved to ensure that duplication of the genome occurs with only minimal risk of mutation induction. One mechanism of genome destabilization is replication fork demise, which can occur when a translocating fork meets a lesion or adduct in the template. Indeed, the collapse of replication forks has been suggested to occur in every replicative cell cycle making this a potentially significant problem for all proliferating cells. The RecQ helicases, which are essential for the maintenance of genome stability, are thought to function during DNA replication. In particular, RecQ helicase mutants display replication defects and have phenotypes consistent with an inability to efficiently reinitiate replication following replication fork demise. Here, we review some current models for how replication fork repair might be effected, and discuss potential roles for RecQ helicases in this process

A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks

DEFF Research Database (Denmark)

Huang, Hongda; Strømme, Caroline B; Saredi, Giulia

2015-01-01

During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase......, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required...... for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling...

High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

Directory of Open Access Journals (Sweden)

Federico Comoglio

2015-05-01

Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

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

Science.gov (United States)

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

2016-01-01

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

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

Science.gov (United States)

Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

2016-01-01

The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

Science.gov (United States)

Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

2015-11-01

Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

Science.gov (United States)

Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E.

2013-01-01

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

NMR structure of the N-terminal domain of the replication initiator protein DnaA

Energy Technology Data Exchange (ETDEWEB)

Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

2007-08-07

DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

Science.gov (United States)

Gan, Haiyun; Yu, Chuanhe; Devbhandari, Sujan; Sharma, Sushma; Han, Junhong; Chabes, Andrei; Remus, Dirk; Zhang, Zhiguo

2017-10-19

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands. Copyright © 2017 Elsevier Inc. All rights reserved.

Pitch Fork

DEFF Research Database (Denmark)

Williams, Peter Leslie; Overholt, Daniel

2017-01-01

Pitch Fork is a prototype of an alternate, actuated digital musical instrument (DMI). It uses 5 infra-red and 4 piezoelectric sensors to control an additive synthesis engine. Iron bars are used as the physical point of contact in interaction with the aim of using this materials natural acoustic p...... properties as a control signal for aspects of the digitally produced sound. This choice of material was also chosen to affect player experience. Sensor readings are relayed to a Macbook via an Arduino Mega. Mappings and audio output signal is carried out with Pure Data Extended....

Did mosasaurs have forked tongues?

NARCIS (Netherlands)

Schulp, Anne S.; Mulder, E. W. A.; Schwenk, K.

Ever since the first mosasaur restorations were published, these extinct marine reptiles have been pictured with either notched, forked or undivided tongues. Here, we present an overview of existing iconography, a review of the previous literature, and we discuss how best to reconstruct tongue form

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

DEFF Research Database (Denmark)

Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien

2012-01-01

but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes...

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

DEFF Research Database (Denmark)

Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia

2007-01-01

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role...

ATM is required for the repair of Topotecan-induced replication-associated double-strand breaks

International Nuclear Information System (INIS)

Köcher, Sabrina; Spies-Naumann, Anja; Kriegs, Malte; Dahm-Daphi, Jochen; Dornreiter, Irena

2013-01-01

Purpose: DNA replication is a promising target for anti-cancer therapies. Therefore, the understanding of replication-associated DNA repair mechanisms is of great interest. One key factor of DNA double-strand break (DSB) repair is the PIK kinase Ataxia-Telangiectasia Mutated (ATM) but it is still unclear whether ATM is involved in the repair of replication-associated DSBs. Here, we focused on the involvement of ATM in homology-directed repair (HDR) of indirect DSBs associated with replication. Material and methods: Experiments were performed using ATM-deficient and -proficient human cells. Replication-associated DSBs were induced with Topotecan (TPT) and compared with γ-irradiation (IR). Cell survival was measured by clonogenic assay. Overall DSB repair and HDR were evaluated by detecting residual γH2AX/53BP1 and Rad51 foci, respectively. Cell cycle distribution was analysed by flow cytometry and protein expression by Western blot. Results: ATM-deficiency leads to enhanced numbers of residual DSBs, resulting in a pronounced S/G2-block and decreased survival upon TPT-treatment. In common with IR, persisting Rad51 foci were detected following TPT-treatment. Conclusions: These results demonstrate that ATM is essentially required for the completion of HR-mediated repair of TPT-induced DSBs formed indirectly at replication forks

Henrys Fork near Ashton, ID (YHEN)

Data.gov (United States)

Department of the Interior — Henrys Fork near Ashton, Idaho (YHEN) Sample Collection: Samples were collected near the USGS stream gage 13046000 (Latitude 44°04'11", Longitude 111°30'38" NAD83)....

Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

International Nuclear Information System (INIS)

Korzhnev, Dmitry M.; Neculai, Dante; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.; Bezsonova, Irina

2016-01-01

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

Energy Technology Data Exchange (ETDEWEB)

Korzhnev, Dmitry M. [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States); Neculai, Dante [Zhejiang University, School of Medicine (China); Dhe-Paganon, Sirano [Dana-Farber Cancer Institute, Department of Cancer Biology (United States); Arrowsmith, Cheryl H. [University of Toronto, Structural Genomics Consortium (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States)

2016-11-15

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

Note: Enhanced energy harvesting from low-frequency magnetic fields utilizing magneto-mechano-electric composite tuning-fork.

Science.gov (United States)

Yang, Aichao; Li, Ping; Wen, Yumei; Yang, Chao; Wang, Decai; Zhang, Feng; Zhang, Jiajia

2015-06-01

A magnetic-field energy harvester using a low-frequency magneto-mechano-electric (MME) composite tuning-fork is proposed. This MME composite tuning-fork consists of a copper tuning fork with piezoelectric Pb(Zr(1-x)Ti(x))O3 (PZT) plates bonded near its fixed end and with NdFeB magnets attached at its free ends. Due to the resonance coupling between fork prongs, the MME composite tuning-fork owns strong vibration and high Q value. Experimental results show that the proposed magnetic-field energy harvester using the MME composite tuning-fork exhibits approximately 4 times larger maximum output voltage and 7.2 times higher maximum power than the conventional magnetic-field energy harvester using the MME composite cantilever.

H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo

Science.gov (United States)

Ejlassi-Lassallette, Aïda; Mocquard, Eloïse; Arnaud, Marie-Claire; Thiriet, Christophe

2011-01-01

While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks. PMID:21118997

Archaeological Investigations on the East Fork of the Salmon River, Custer County, Idaho.

Science.gov (United States)

1984-01-01

coniferous environment in addition to pine marten (Martes americana), red squirrel (Tamiasciurus hudsonicus), porcupine (Erithizon dorsatum), mountain vole...can be seen in small herds throughout the East Fork valley from the Salmon River to Big Boulder Creek. Two bands of Rocky Mountain bighorn sheep...utilize the Challis Planning Unit, one on the East Fork and the other in the Birch Creek area. The East Fork herd is comprised of approximately 50-70

Structural design of a composite bicycle fork

International Nuclear Information System (INIS)

Baldissera, Paolo; Delprete, Cristiana

2014-01-01

Highlights: • Case study about composite bicycle fork design. • Special requirements for a Student Team project. • FE model to evaluate stiffness, strength and potential failure modes. • Comparison of two manufacturing approaches. • FE model stiffness validation on the manufactured fork. - Abstract: Despite the wide literature on the mechanical behaviour of carbon/epoxy composites, it is rare to find practical methodological approaches in finite element design of structural components made by laminate layup. Through the case study of a special bicycle fork needed in a Student Team prototype, this paper proposes a simplified methodology as starting point for educational and manufacturing purposes. In order to compare two manufacturing solutions in terms of stiffness, strength and failure mode, a numerical model was implemented. Since the project requirements imposed to avoid standard destructive testing, the model validation was based on a posteriori linear stiffness comparison with the manufactured component. The slight discrepancies between experimental and numerical results were discussed in order to check their origin and to assess the reliability of the model. The overall methodology, even if complain with only a part of the safety standard requirements, shows to be reliable enough and can be the basis for further extension and refinement

77 FR 39675 - Wallowa-Whitman National Forest, Baker County, OR; North Fork Burnt River Mining

Science.gov (United States)

2012-07-05

...-Whitman National Forest, Baker County, OR; North Fork Burnt River Mining AGENCY: Forest Service, USDA... North Fork Burnt River Mining Record of Decision will replace and supercede the 2004 North Fork Burnt River Mining Record of Decision only where necessary to address the inadequacies identified by the court...

UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

Directory of Open Access Journals (Sweden)

Kelley N. Newton

2012-01-01

Full Text Available UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

Science.gov (United States)

Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

1990-02-25

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

Theoretical models for the regulation of DNA replication in fast-growing bacteria

Science.gov (United States)

Creutziger, Martin; Schmidt, Mischa; Lenz, Peter

2012-09-01

Growing in always changing environments, Escherichia coli cells are challenged by the task to coordinate growth and division. In particular, adaption of their growth program to the surrounding medium has to guarantee that the daughter cells obtain fully replicated chromosomes. Replication is therefore to be initiated at the right time, which is particularly challenging in media that support fast growth. Here, the mother cell initiates replication not only for the daughter but also for the granddaughter cells. This is possible only if replication occurs from several replication forks that all need to be correctly initiated. Despite considerable efforts during the last 40 years, regulation of this process is still unknown. Part of the difficulty arises from the fact that many details of the relevant molecular processes are not known. Here, we develop a novel theoretical strategy for dealing with this general problem: instead of analyzing a single model, we introduce a wide variety of 128 different models that make different assumptions about the unknown processes. By comparing the predictions of these models we are able to identify the key quantities that allow the experimental discrimination of the different models. Analysis of these quantities yields that out of the 128 models 94 are not consistent with available experimental data. From the remaining 34 models we are able to conclude that mass growth and DNA replication need either to be truly coupled, by coupling DNA replication initiation to the event of cell division, or to the amount of accumulated mass. Finally, we make suggestions for experiments to further reduce the number of possible regulation scenarios.

Replication of cultured lung epithelial cells

International Nuclear Information System (INIS)

Guzowski, D.; Bienkowski, R.

1986-01-01

The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to ( 3 H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems

Replication-independent chromatin loading of Dnmt1 during G2 and M phases

Science.gov (United States)

Easwaran, Hariharan P; Schermelleh, Lothar; Leonhardt, Heinrich; Cardoso, M Cristina

2004-01-01

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation. PMID:15550930

Performance Evaluation of the New Fork-Absorbers of RSG-GAS Control Rod

International Nuclear Information System (INIS)

Slamet Wiranto; Purwadi; Arif Hidayat; Agus Sanjaya

2012-01-01

During the operation of RSG-GAS reactor, it has been replaced 8 fork-absorber by the new absorber from PT. Batan Teknologi. After almost 5 years under utilization it is important to be evaluated to determine the physical condition and its performance, which is still in good condition and functioning according to the requirements of its operations. The evaluation has been carried out by studying and analyzing the data of the fork-absorber utilization in the the reactor core. The fork absorber data consist of visual inspection, control rod drop time measurement and control rod reactivity and safety margin measurement for each operation cycle. Through the observation up to date with the operating cycle of 79, could be concluded that the fork-absorber condition is still good, and has ability, to support the operation until ± 660 MWD/cycle, which is characterized by obtaining the value of ρ-excess is sufficient for operation, with a large safety margin. (author)

Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

Science.gov (United States)

Nakazaki, Yuta; Tsuyama, Takashi; Azuma, Yutaro; Takahashi, Mikiko; Tada, Shusuke

2017-09-02

The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of the temporal program of replication initiation in yeast chromosomes.

Science.gov (United States)

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

1995-01-01

The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase. In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an internal position on chromosome XIV. Four origins, spanning approximately 140 kb, initiate replication in the second half of S phase. At least two of these internal origins maintain their late replication time on circular plasmids. Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation. Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these 'late' elements. We report here the development of two new assays for determining replication time. The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork. The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination. This non-replicating fragment acts as an internal control for copy number within and between samples. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA. With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

Science.gov (United States)

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

2016-01-01

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

Characterizing the Final Steps of Chromosomal Replication at the Single-molecule Level in the Model System Escherichia coli

KAUST Repository

Elshenawy, Mohamed M.

2015-12-01

In the circular Escherichia coli chromosome, two replisomes are assembled at the unique origin of replication and drive DNA synthesis in opposite directions until they meet in the terminus region across from the origin. Despite the difference in rates of the two replisomes, their arrival at the terminus is synchronized through a highly specialized system consisting of the terminator protein (Tus) bound to the termination sites (Ter). This synchronicity is mediated by the polarity of the Tus−Ter complex that stops replisomes from one direction (non-permissive face) but not the other (permissive face). Two oppositely oriented clusters of five Tus–Ters that each block one of the two replisomes create a “replication fork trap” for the first arriving replisome while waiting for the late arriving one. Despite extensive biochemical and structural studies, the molecular mechanism behind Tus−Ter polar arrest activity remained controversial. Moreover, none of the previous work provided answers for the long-standing discrepancy between the ability of Tus−Ter to permanently stop replisomes in vitro and its low efficiency in vivo. Here, I spearheaded a collaborative project that combined single-molecule DNA replication assays, X-ray crystallography and binding studies to provide a true molecular-level understanding of the underlying mechanism of Tus−Ter polar arrest activity. We showed that efficiency of Tus−Ter is determined by a head-to-head kinetic competition between rate of strand separation by the replisome and rate of rearrangement of Tus−Ter interactions during the melting of the first 6 base pairs of Ter. This rearrangement maintains Tus’s strong grip on the DNA and stops the advancing replisome from breaking into Tus−Ter central interactions, but only transiently. We further showed how this kinetic competition functions within the context of two mechanisms to impose permanent fork stoppage. The rate-dependent fork arrest activity of Tus�

Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress

Czech Academy of Sciences Publication Activity Database

Maya-Mendoza, A.; Ostrakova, J.; Košař, Martin; Hall, A.; Duskova, P.; Mistrik, M.; Merchut-Maya, J.M.; Hodný, Zdeněk; Bartkova, J.; Christensen, C.; Bartek, Jiří

2015-01-01

Roč. 9, č. 3 (2015), s. 601-616 ISSN 1574-7891 R&D Projects: GA ČR GA13-17555S; GA MŠk(CZ) LO1304 Grant - others:Danish Council for Independent Research(DK) DFF- 1331-00262; Lundbeck Foundation(DK) R93-A8990 Institutional support: RVO:68378050 Keywords : Myc * Ras * Replication stress * DNA fork progression * Energy metabolism * DNA damage response Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.367, year: 2015

New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

Science.gov (United States)

Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

2015-05-01

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory

Reduced fuel consumption for fork-lift trucks with hydrostatic transmission

Energy Technology Data Exchange (ETDEWEB)

Abels, T

1983-05-01

Cost calculations for a 3,5-t diesel fork lifter done on the basis of VDI 2695 shows, that fuel costs account only for a small part of the operating costs despite the price increase for diesel fuel. Fork lifters with disk-cam controlled primary/secondary adjusted hydrostatic transmission used less fuel than was indicated in the VDI-guideline. Fuel consumption could further be reduced by an optimized hydraulic adjustment together with a precisely harmonized engine speed adjustment. Annual cost savings are considerable.

USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication.

Science.gov (United States)

Hernández-Pérez, Santiago; Cabrera, Elisa; Amoedo, Hugo; Rodríguez-Acebes, Sara; Koundrioukoff, Stephane; Debatisse, Michelle; Méndez, Juan; Freire, Raimundo

2016-10-01

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Final Environmental Assessment: Installation of Digital Airport Surveillance Radar at Grand Forks Air Force Base, North Dakota

Science.gov (United States)

2011-05-11

Grand Forks AFB public web site. Notices of Availability were published in the Grand Forks Herald on 10 Mar 2011 and on the Grand Forks AFB web site from...Squadron (319th) FTA Fire Training Area GATR Ground-Air Transmit Receive GFAFB Grand Forks Air Force Base GHG Greenhouse Gas Hz Hertz IEEE Institute of...feet west of the closed/capped ERP Site FT-02, the Fire Training Area/Old Sanitary Landfill Area ( FTA /OSLA), which encompasses 28 acres, five of

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.

Science.gov (United States)

Alver, Robert C; Chadha, Gaganmeet Singh; Gillespie, Peter J; Blow, J Julian

2017-03-07

Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Replicate periodic windows in the parameter space of driven oscillators

Energy Technology Data Exchange (ETDEWEB)

Medeiros, E.S., E-mail: esm@if.usp.br [Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo (Brazil); Souza, S.L.T. de [Universidade Federal de Sao Joao del-Rei, Campus Alto Paraopeba, Minas Gerais (Brazil); Medrano-T, R.O. [Departamento de Ciencias Exatas e da Terra, Universidade Federal de Sao Paulo, Diadema, Sao Paulo (Brazil); Caldas, I.L. [Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo (Brazil)

2011-11-15

Highlights: > We apply a weak harmonic perturbation to control chaos in two driven oscillators. > We find replicate periodic windows in the driven oscillator parameter space. > We find that the periodic window replication is associated with the chaos control. - Abstract: In the bi-dimensional parameter space of driven oscillators, shrimp-shaped periodic windows are immersed in chaotic regions. For two of these oscillators, namely, Duffing and Josephson junction, we show that a weak harmonic perturbation replicates these periodic windows giving rise to parameter regions correspondent to periodic orbits. The new windows are composed of parameters whose periodic orbits have the same periodicity and pattern of stable and unstable periodic orbits already existent for the unperturbed oscillator. Moreover, these unstable periodic orbits are embedded in chaotic attractors in phase space regions where the new stable orbits are identified. Thus, the observed periodic window replication is an effective oscillator control process, once chaotic orbits are replaced by regular ones.

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–MMS22L^{1, 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.

Oriented Onion Sowing by a Forked-Roller Sowing Unit

Directory of Open Access Journals (Sweden)

Aleksandr G.

2018-03-01

Full Text Available Introduction: The existing sowing machines do not provide a single feeding of the bulbs with a planting (sowing unit that leads to a violation of the agrotechnical requirements of planting bulbs. It is necessary to search new solutions to preserve the position of the bulbs in the furrow with the bottom down and their regularly spaced distribution. Materials and Methods: The article presents the design for a prototype for a planting machine equipped with a forked-roller sowing unit for orienting the onion-sowing into a furrow. Testing the forked-roller sowing unit were carried out on a flat area where the physical and mechanical properties of the soil were determined on the days of sowing, and the indices of the quality of the onion-sowing were determined. The study of the effect of the sowing machine speed on the quality of the onion-seed bulb landing was determined by the change in the translational speed of the sowing unit in the range of 0.8 m/s to 1.2 m/s with a variation interval of 0.1 m/s. The indicators of the quality of the planting of the bulbs were determined by the opening of the closed furrow. The results of laboratory-field studies of the planting machine prototype are presented. Results: The results of laboratory-field studies of a planting machine equipped with a forked-roller sowing unit for planting onion bulbs are presented. The optimal technological parameters are determined experimentally. It was determined the number of bulbs that are for up is 51 % and the regularity of planting by the forked-roller sowing unit – 79 %. These figures are provided at the forward speed of the planting machine VM = 0.9–1.0 m/s, the height of the fall of the bulb HA = 0.12 m, and the rotation frequency of the landing drum nБ = 0.47 c-1. Discussion and Conclusions: The use of a forked-roller sowing unit makes it possible to increase the proportion of onions planted by bottom down by 200 %, and the uniformity of planting bulbs by 19 %, in

Simple force balance accelerometer/seismometer based on a tuning fork displacement sensor

International Nuclear Information System (INIS)

Stuart-Watson, D.; Tapson, J.

2004-01-01

Seismometers and microelectromechanical system accelerometers use the force-balance principle to obtain measurements. In these instruments the displacement of a mass object by an unknown force is sensed using a very high-resolution displacement sensor. The position of the object is then stabilized by applying an equal and opposite force to it. The magnitude of the stabilizing force is easily measured, and is assumed to be equivalent to the unknown force. These systems are critically dependent on the displacement sensor. In this article we use a resonant quartz tuning fork as the sensor. The tuning fork is operated so that its oscillation is lightly damped by the proximity of the movable mass object. Changes in the position of the mass object cause changes in the phase of the fork's resonance; this is used as the feedback variable in controlling the mass position. We have developed an acceleration sensor using this principle. The mass object is a piezoelectric bimorph diaphragm which is anchored around its perimeter, allowing direct electronic control of the displacement of its center. The tuning fork is brought very close to the diaphragm center, and is connected into a self-oscillating feedback circuit which has phase and amplitude as outputs. The diaphragm position is adjusted by a feedback loop, using phase as the feedback variable, to keep it in a constant position with respect to the tuning fork. The measured noise for this sensor is approximately 10.0 mg in a bandwidth of 100 Hz, which is substantially better than commercial systems of equivalent cost and size

Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

Science.gov (United States)

Dembowski, Jill A; DeLuca, Neal A

2015-05-01

Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and

The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress.

Science.gov (United States)

Zhang, You-Wei; Brognard, John; Coughlin, Chris; You, Zhongsheng; Dolled-Filhart, Marisa; Aslanian, Aaron; Manning, Gerard; Abraham, Robert T; Hunter, Tony

2009-08-28

ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.

Development of the Pintle Release Fork Mechanism

International Nuclear Information System (INIS)

BOGER, R.M.; DALE, R.

1999-01-01

An improved method of attachment of the pintle to the piston in the universal sampler is being developed. The mechanism utilizes a forked release disk which captures two balls in a cavity formed by a hole in the piston and a groove in the pintle rod

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

Directory of Open Access Journals (Sweden)

Bonita J Brewer

2015-12-01

Full Text Available DNA replication errors are a major driver of evolution--from single nucleotide polymorphisms to large-scale copy number variations (CNVs. Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model--Origin-Dependent Inverted-Repeat Amplification (ODIRA-proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error-the ligation of leading and lagging nascent strands to create "closed" forks-can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent--a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

Science.gov (United States)

Brewer, Bonita J; Payen, Celia; Di Rienzi, Sara C; Higgins, Megan M; Ong, Giang; Dunham, Maitreya J; Raghuraman, M K

2015-12-01

DNA replication errors are a major driver of evolution--from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model--Origin-Dependent Inverted-Repeat Amplification (ODIRA)-proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error-the ligation of leading and lagging nascent strands to create "closed" forks-can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent--a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial

Von Economo Neurons and Fork Cells: A Neurochemical Signature Linked to Monoaminergic Function.

Science.gov (United States)

Dijkstra, Anke A; Lin, Li-Chun; Nana, Alissa L; Gaus, Stephanie E; Seeley, William W

2018-01-01

The human anterior cingulate and frontoinsular cortices are distinguished by 2 unique Layer 5 neuronal morphotypes, the von Economo neurons (VENs) and fork cells, whose biological identity remains mysterious. Insights could impact research on diverse neuropsychiatric diseases to which these cells have been linked. Here, we leveraged the Allen Brain Atlas to evaluate mRNA expression of 176 neurotransmitter-related genes and identified vesicular monoamine transporter 2 (VMAT2), gamma-aminobutyric acid (GABA) receptor subunit θ (GABRQ), and adrenoreceptor α-1A (ADRA1A) expression in human VENs, fork cells, and a minority of neighboring Layer 5 neurons. We confirmed these results using immunohistochemistry or in situ hybridization. VMAT2 and GABRQ expression was absent in mouse cerebral cortex. Although VMAT2 is known to package monoamines into synaptic vesicles, in VENs and fork cells its expression occurs in the absence of monoamine-synthesizing enzymes or reuptake transporters. Thus, VENs and fork cells may possess a novel, uncharacterized mode of cortical monoaminergic function that distinguishes them from most other mammalian Layer 5 neurons. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Homologous recombination in mammalian cells: effect of p53 and Bcl-2 proteins, replication inhibition and ionizing radiations

International Nuclear Information System (INIS)

Saintigny, Yannick

1999-01-01

The control of cell cycle, associated with the mechanisms of replication, DNA repair/recombination allows the cells to maintain their genetic integrity. The p53 protein ensures the control of G1/S transition. Its inactivation would allow to initial replication on damaged matrix and lead to the block of replication forks followed by DNA strand breaks, good substrates for recombination. This work shows that the expression of mutant p53 protein stimulates both spontaneous and radio-induced homologous recombination, independently of the control of cell cycle. Moreover, the use of a set of replication inhibitors show that inhibition of the replication elongation stimulates recombination more strongly than the initiation inhibition. Replication arrest by these inhibitors also significantly increases the number of DNA strand breaks. These results highlighted a point of action of p53 protein on the ultimate stages of the homologous recombination mechanism. Lastly, the expression of Bcl-2 protein inhibits apoptosis and increases survival, but specifically inhibits conservative recombination, after radiation as well as in absence of apoptotic stress. The extinction of this mechanism of DNA repair is associated with an increase of mutagenesis. Taken together, these results allow ta consider the maintenance of the genetic stability as a cellular network involving different pathways. A multiple stages model for tumoral progression can be deduced. (author) [fr

Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication.

Science.gov (United States)

Bj Rås, Karine Ø; Sousa, Mirta M L; Sharma, Animesh; Fonseca, Davi M; S Gaard, Caroline K; Bj Rås, Magnar; Otterlei, Marit

2017-08-21

Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Coefficient of the Voltage Induced Frequency Shift Measurement on a Quartz Tuning Fork

Directory of Open Access Journals (Sweden)

Yubin Hou

2014-11-01

Full Text Available We have measured the coefficient of the voltage induced frequency shift (VIFS of a 32.768 KHz quartz tuning fork. Three vibration modes were studied: one prong oscillating, two prongs oscillating in the same direction, and two prongs oscillating in opposite directions. They all showed a parabolic dependence of the eigen-frequency shift on the bias voltage applied across the fork, due to the voltage-induced internal stress, which varies as the fork oscillates. The average coefficient of the VIFS effect is as low as several hundred nano-Hz per millivolt, implying that fast-response voltage-controlled oscillators and phase-locked loops with nano-Hz resolution can be built.

Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

International Nuclear Information System (INIS)

Tao Weitao; Budd, Martin; Campbell, Judith L.

2003-01-01

We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Δ double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Δ alone. However, surprisingly, the dna2-2 sgs1Δ lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Δ lethality is only partially suppressed by deletion of rad51Δ. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones

Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

Energy Technology Data Exchange (ETDEWEB)

Tao Weitao; Budd, Martin; Campbell, Judith L

2003-11-27

We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1{delta} double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1{delta} alone. However, surprisingly, the dna2-2 sgs1{delta} lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1{delta} lethality is only partially suppressed by deletion of rad51{delta}. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

Theoretical model and optimization of a novel temperature sensor based on quartz tuning fork resonators

International Nuclear Information System (INIS)

Xu Jun; You Bo; Li Xin; Cui Juan

2007-01-01

To accurately measure temperatures, a novel temperature sensor based on a quartz tuning fork resonator has been designed. The principle of the quartz tuning fork temperature sensor is that the resonant frequency of the quartz resonator changes with the variation in temperature. This type of tuning fork resonator has been designed with a new doubly rotated cut work at flexural vibration mode as temperature sensor. The characteristics of the temperature sensor were evaluated and the results sufficiently met the target of development for temperature sensor. The theoretical model for temperature sensing has been developed and built. The sensor structure was analysed by finite element method (FEM) and optimized, including tuning fork geometry, tine electrode pattern and the sensor's elements size. The performance curve of output versus measured temperature is given. The results from theoretical analysis and experiments indicate that the sensor's sensitivity can reach 60 ppm 0 C -1 with the measured temperature range varying from 0 to 100 0 C

76 FR 46721 - Salmon-Challis National Forest, ID; Upper North Fork HFRA Ecosystem Restoration Project...

Science.gov (United States)

2011-08-03

...-Challis National Forest, ID; Upper North Fork HFRA Ecosystem Restoration Project Environmental Impact... improve the health of the ecosystem and reach the desired future condition. DATES: Comments concerning the... Ecosystem Restoration Project EIS, P.O. Box 180, 11 Casey Rd., North Fork, ID 83466. Comments may also be...

Spent-fuel verification with the Los Alamos fork detector

International Nuclear Information System (INIS)

Rinard, P.M.; Bosler, G.E.

1987-01-01

The Los Alamos fork detector for the verification of spent-fuel assemblies has generated precise, reproducible data. The data analyses have now evolved to the point of placing tight restrictions on a diverter's actions

The effectiveness of front fork systems at damping accelerations during isolated aspects specific to cross-country mountain biking.

Science.gov (United States)

Macdermid, Paul W; Miller, Matthew C; Fink, Philip W; Stannard, Stephen R

2017-11-01

Cross-country mountain bike suspension reportedly enhances comfort and performance through reduced vibration and impact exposure. This study analysed the effectiveness of three different front fork systems at damping accelerations during the crossing of three isolated obstacles (stairs, drop, and root). One participant completed three trials on six separate occasions in a randomised order using rigid, air-sprung, and carbon leaf-sprung forks. Performance was determined by time to cross obstacles, while triaxial accelerometers quantified impact exposure and damping response. Results identified significant main effect of fork type for performance time (p < 0.05). The air-sprung and leaf-sprung forks were significantly slower than the rigid forks for the stairs (p < 0.05), while air-sprung suspension was slower than the rigid for the root protocol (p < 0.05). There were no differences for the drop protocol (p < 0.05). Rigid forks reduced overall exposure (p < 0.05), specifically at the handlebars for the stairs and drop trials. More detailed analysis presented smaller vertical accelerations at the handlebar for air-sprung and leaf-sprung forks on the stairs (p < 0.05), and drop (p < 0.05) but not the root. As such, it appears that the suspension systems tested were ineffective at reducing overall impact exposure at the handlebar during isolated aspects of cross-country terrain features which may be influenced to a larger extent by rider technique.

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

DEFF Research Database (Denmark)

Di Marco, Stefano; Hasanova, Zdenka; Kanagaraj, Radhakrishnan

2017-01-01

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent...... on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain...

InterProScan Result: FS906632 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available 8 T IPR012923 Replication fork protection component Swi3 Cellular Component: nucleus (GO:0005634)|Biological... Process: response to DNA damage stimulus (GO:0006974)|Biological Process: cell cycle (GO:0007049)|Biological Process: replication fork protection (GO:0048478) ...

Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication.

Science.gov (United States)

Ramadan, Kristijan; Halder, Swagata; Wiseman, Katherine; Vaz, Bruno

2017-02-01

Genome amplification (DNA synthesis) is one of the most demanding cellular processes in all proliferative cells. The DNA replication machinery (also known as the replisome) orchestrates genome amplification during S-phase of the cell cycle. Genetic material is particularly vulnerable to various events that can challenge the replisome during its assembly, activation (firing), progression (elongation) and disassembly from chromatin (termination). Any disturbance of the replisome leads to stalling of the DNA replication fork and firing of dormant replication origins, a process known as DNA replication stress. DNA replication stress is considered to be one of the main causes of sporadic cancers and other pathologies related to tissue degeneration and ageing. The mechanisms of replisome assembly and elongation during DNA synthesis are well understood. However, once DNA synthesis is complete, the process of replisome disassembly, and its removal from chromatin, remains unclear. In recent years, a growing body of evidence has alluded to a central role in replisome regulation for the ubiquitin-dependent protein segregase p97, also known as valosin-containing protein (VCP) in metazoans and Cdc48 in lower eukaryotes. By orchestrating the spatiotemporal turnover of the replisome, p97 plays an essential role in DNA replication. In this review, we will summarise our current knowledge about how p97 controls the replisome from replication initiation, to elongation and finally termination. We will also further examine the more recent findings concerning the role of p97 and how mutations in p97 cofactors, also known as adaptors, cause DNA replication stress induced genomic instability that leads to cancer and accelerated ageing. To our knowledge, this is the first comprehensive review concerning the mechanisms involved in the regulation of DNA replication by p97.

76 FR 35909 - Temporary Concession Contract for Big South Fork National Recreation Area, TN/KY

Science.gov (United States)

2011-06-20

... Recreation Area, TN/KY. SUMMARY: Pursuant to 36 CFR 51.24, public notice is hereby given that the National... Concession Contract for Big South Fork National Recreation Area, TN/KY AGENCY: National Park Service... services within Big South Fork National Recreation Area, Tennessee and Kentucky, for a term not to exceed 3...

Flood-inundation maps for the East Fork White River at Columbus, Indiana

Science.gov (United States)

Lombard, Pamela J.

2013-01-01

Digital flood-inundation maps for a 5.4-mile reach of the East Fork White River at Columbus, Indiana, from where the Flatrock and Driftwood Rivers combine to make up East Fork White River to just upstream of the confluence of Clifty Creek with the East Fork White River, were created by the U.S. Geological Survey (USGS) in cooperation with the Indiana Department of Transportation. The inundation maps, which can be accessed through the USGS Flood Inundation Mapping Science Web site at http://water.usgs.gov/osw/flood_inundation, depict estimates of the areal extent of flooding corresponding to selected water levels (stages) at USGS streamgage 03364000, East Fork White River at Columbus, Indiana. Current conditions at the USGS streamgage may be obtained on the Internet from the USGS National Water Information System (http://waterdata.usgs.gov/in/nwis/uv/?site_no=03364000&agency_cd=USGS&). The National Weather Service (NWS) forecasts flood hydrographs for the East Fork White River at Columbus, Indiana at their Advanced Hydrologic Prediction Service (AHPS) flood warning system Website (http://water.weather.gov/ahps/), that may be used in conjunction with the maps developed in this study to show predicted areas of flood inundation. In this study, flood profiles were computed for the stream reach by means of a one-dimensional step-backwater model. The hydraulic model was calibrated by using the most current stage-discharge relation at USGS streamgage 03364000, East Fork White River at Columbus, Indiana. The calibrated hydraulic model was then used to determine 15 water-surface profiles for flood stages at 1-foot (ft) intervals referenced to the streamgage datum and ranging from bankfull to approximately the highest recorded water level at the streamgage. The simulated water-surface profiles were then combined with a geographic information system digital elevation model (derived from Light Detection and Ranging (LiDAR) data), having a 0.37-ft vertical accuracy and a 1.02 ft

Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

Directory of Open Access Journals (Sweden)

Jill A Dembowski

2015-05-01

Full Text Available Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics

The Sound Field around a Tuning Fork and the Role of a Resonance Box

Science.gov (United States)

Bogacz, Bogdan F.; Pedziwiatr, Antoni T.

2015-01-01

Atypical two-tine tuning fork is barely audible when held vibrating at an arm's length. It is enough, however, to touch its base to a table or, better, to a resonance box and the emitted sound becomes much louder. An inquiring student may pose questions: (1) Why is a bare tuning fork such a weak emitter of sound? (2) What is the role of the…

Burnup verification tests with the FORK measurement system-implementation for burnup credit

International Nuclear Information System (INIS)

Ewing, R.I.

1994-01-01

Verification measurements may be used to help ensure nuclear criticality safety when burnup credit is applied to spent fuel transport and storage systems. The FORK system measures the passive neutron and gamma-ray emission from spent fuel assemblies while in the storage pool. It was designed at Los Alamos National Laboratory for the International Atomic Energy Agency safeguards program and is well suited to verify burnup and cooling time records at commercial Pressurized Water Reactor (PWR) sites. This report deals with the application of the FORK system to burnup credit operations

Linker-dependent Junction Formation Probability in Single-Molecule Junctions

Energy Technology Data Exchange (ETDEWEB)

Yoo, Pil Sun; Kim, Taekyeong [HankukUniversity of Foreign Studies, Yongin (Korea, Republic of)

2015-01-15

We compare the junction formation probabilities of single-molecule junctions with different linker molecules by using a scanning tunneling microscope-based break-junction technique. We found that the junction formation probability varies as SH > SMe > NH2 for the benzene backbone molecule with different types of anchoring groups, through quantitative statistical analysis. These results are attributed to different bonding forces according to the linker groups formed with Au atoms in the electrodes, which is consistent with previous works. Our work allows a better understanding of the contact chemistry in the metal.molecule junction for future molecular electronic devices.

Identification of proteins that may directly interact with human RPA.

Science.gov (United States)

Nakaya, Ryou; Takaya, Junichiro; Onuki, Takeshi; Moritani, Mariko; Nozaki, Naohito; Ishimi, Yukio

2010-11-01

RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.

Division of Labor

KAUST Repository

Oke, Muse; Zaher, Manal S.; Hamdan, Samir

2014-01-01

The first assignment of DNA polymerases at the eukaryotic replication fork was possible after the in vitro reconstitution of the simian virus 40 (SV40) replication system. In this system, DNA polymerase α (Pol α) provides both leading and lagging strands with RNA-DNA primers that are extended by DNA polymerase δ (Pol δ). Extrapolating the architecture of the replication fork from the SV40 model system to an actual eukaryotic cell has been challenged by the discovery of a third DNA polymerase in Saccharomyces cerevisiae, DNA polymerase ε (Pol ε). A division of labor has been proposed for the eukaryotic replication fork whereby Pol ε replicates the leading strand and Pol δ replicates the lagging strand. However, an alternative model of unequal division of labor in which Pol δ can still participate in leading-strand synthesis is plausible.

Division of Labor

KAUST Repository

Oke, Muse

2014-09-12

The first assignment of DNA polymerases at the eukaryotic replication fork was possible after the in vitro reconstitution of the simian virus 40 (SV40) replication system. In this system, DNA polymerase α (Pol α) provides both leading and lagging strands with RNA-DNA primers that are extended by DNA polymerase δ (Pol δ). Extrapolating the architecture of the replication fork from the SV40 model system to an actual eukaryotic cell has been challenged by the discovery of a third DNA polymerase in Saccharomyces cerevisiae, DNA polymerase ε (Pol ε). A division of labor has been proposed for the eukaryotic replication fork whereby Pol ε replicates the leading strand and Pol δ replicates the lagging strand. However, an alternative model of unequal division of labor in which Pol δ can still participate in leading-strand synthesis is plausible.

A watershed's response to logging and roads: South Fork of Caspar Creek, California, 1967-1976

Science.gov (United States)

Raymond M. Rice; Forest B. Tilley; Patricia A. Datzman

1979-01-01

The effect of logging and roadbuilding on erosion and sedimentation are analyzed by comparing the North Fork and South Fork of Caspar Creek, in northern California. Increased sediment production during the 4 years after road construction, was 326 cu yd/sq mi/yr—80 percent greater than that predicted by the predisturbance regression analysis. The average...

A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

Science.gov (United States)

Pohl, Thomas J; Kolor, Katherine; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

2013-11-06

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid

Directory of Open Access Journals (Sweden)

Aiken Christopher

2004-06-01

Full Text Available Abstract Background Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells. Results Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug. Conclusions These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.

33 CFR 165.552 - Security Zone; Oyster Creek Generation Station, Forked River, Ocean County, New Jersey.

Science.gov (United States)

2010-07-01

... Generation Station, Forked River, Ocean County, New Jersey. 165.552 Section 165.552 Navigation and Navigable... Coast Guard District § 165.552 Security Zone; Oyster Creek Generation Station, Forked River, Ocean... part. (2) No person or vessel may enter or navigate within this security zone unless authorized to do...

An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

Science.gov (United States)

Perez-Arnaiz, Patricia; Kaplan, Daniel L

2016-11-20

Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanically stable tuning fork sensor with high quality factor for the atomic force microscope.

Science.gov (United States)

Kim, Kwangyoon; Park, Jun-Young; Kim, K B; Lee, Naesung; Seo, Yongho

2014-01-01

A quartz tuning fork was used instead of cantilever as a force sensor for the atomic force microscope. A tungsten tip was made by electrochemical etching from a wire of 50 µm diameter. In order to have mechanical stability of the tuning fork, it was attached on an alumina plate. The tungsten tip was attached on the inside end of a prong of a tuning fork. The phase shift was used as a feedback signal to control the distance between the tip and sample, and the amplitude was kept constant using a lock-in amplifier and a homemade automatic gain controller. Due to the mechanical stability, the sensor shows a high quality factor (∼10(3)), and the image quality obtained with this sensor was equivalent to that of the cantilever-based AFM. © 2014 Wiley Periodicals, Inc.

Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells.

Science.gov (United States)

Hodroj, Dana; Serhal, Kamar; Maiorano, Domenico

2017-09-03

The DEAD-box Helicase 19 (Ddx19) gene codes for an RNA helicase involved in both mRNA (mRNA) export from the nucleus into the cytoplasm and in mRNA translation. In unperturbed cells, Ddx19 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore. Here we review recent findings related to an additional Ddx19 function in the nucleus in resolving RNA:DNA hybrids (R-loops) generated during collision between transcription and replication, and upon DNA damage. Activation of a DNA damage response pathway dependent upon the ATR kinase, a major regulator of replication fork progression, stimulates translocation of the Ddx19 protein from the cytoplasm into the nucleus. Only nuclear Ddx19 is competent to resolve R-loops, and down regulation of Ddx19 expression induces DNA double strand breaks only in proliferating cells. Overall these observations put forward Ddx19 as an important novel mediator of the crosstalk between transcription and replication.

Uncertainty Quantification of Fork Detector Measurements from Spent Fuel Loading Campaigns

International Nuclear Information System (INIS)

Vaccaro, S.; De Baere, P.; Schwalbach, P.; Gauld, I.; Hu, J.

2015-01-01

With increasing activities at the end of the fuel cycle, the requirements for the verification of spent nuclear fuel for safeguards purposes are continuously growing. In the European Union we are experiencing a dramatic increase in the number of cask loadings for interim dry storage. This is caused by the progressive shut-down of reactors, related to facility ageing but also due to politically motivated phase-out of nuclear power. On the other hand there are advanced plans for the construction of encapsulation plants and geological repositories. The cask loading or the encapsulation process will provide the last occasion to verify the spent fuel assemblies. In this context, Euratom and the US DOE have carried out a critical review of the widely used Fork measurements method of irradiated assemblies. The Nuclear Safeguards directorates of the European Commission's Directorate General for Energy and Oak Ridge National Laboratory have collaborated to improve the Fork data evaluation process and simplify its use for inspection applications. Within the Commission's standard data evaluation package CRISP, we included a SCALE/ORIGEN-based irradiation and depletion simulation of the measured assembly and modelled the fork transfer function to calculate expected count rates based on operator's declarations. The complete acquisition and evaluation process has been automated to compare expected (calculated) with measured count rates. This approach allows a physics-based improvement of the data review and evaluation process. At the same time the new method provides the means for better measurement uncertainty quantification. The present paper will address the implications of the combined approach involving measured and simulated data to the quantification of measurement uncertainty and the consequences of these uncertainties in the possible use of the Fork detector as a partial defect detection method. (author)

Triptycene: A Nucleic Acid Three-Way Junction Binder Scaffold

Science.gov (United States)

Yoon, Ina

Nucleic acids play a critical role in many biological processes such as gene regulation and replication. The development of small molecules that modulate nucleic acids with sequence or structure specificity would provide new strategies for regulating disease states at the nucleic acid level. However, this remains challenging mainly because of the nonspecific interactions between nucleic acids and small molecules. Three-way junctions are critical structural elements of nucleic acids. They are present in many important targets such as trinucleotide repeat junctions related to Huntington's disease, a temperature sensor sigma32 in E. coli, Dengue virus, and HIV. Triptycene-derived small molecules have been shown to bind to nucleic acid three-way junctions, resulting from their shape complementary. To develop a better understanding of designing molecules for targeting different junctions, a rapid screening of triptycene-based small molecules is needed. We envisioned that the installation of a linker at C9 position of the bicyclic core would allow for a rapid solid phase diversification. To achieve this aim, we synthesized 9-substituted triptycene scaffolds by using two different synthetic routes. The first synthetic route installed the linker from the amidation reaction between carboxylic acid at C9 position of the triptycene and an amine linker, beta-alanine ethyl ester. This new 9-substituted triptycene scaffold was then attached to a 2-chlorotrityl chloride resin for solid-phase diversification. This enabled a rapid diversification and an easy purification of mono-, di-, and tri-peptide triptycene derivatives. The binding affinities of these compounds were investigated towards a (CAG)˙(CTG) trinucleotide repeat junction. In the modified second synthetic route, we utilized a combined Heck coupling/benzyne Diels-Alder strategy. This improved synthetic strategy reduced the number of steps and total reaction times, increased the overall yield, improved solubilities of

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress.

Directory of Open Access Journals (Sweden)

Raymond Buser

2016-02-01

Full Text Available Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4, but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101(Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101(Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS complex at stalled forks.

Sediment transport and storage in North Fork Caspar Creek, Mendocino County, California: water years 1980-1988

Science.gov (United States)

Michael Brent Napolitano

1996-01-01

Abstract - The old-growth redwood forest of North Fork Caspar Creek was clear-cut between 1864 and 1904. Previous research on logging-related changes in suspended sediment and streamflow would suggest that North Fork Caspar Creek has recovered from historical logging (Rice et al., 1979; Ziemer, 1981); research on the influence of large woody debris (LWD) on channel...

Chemical bond imaging using higher eigenmodes of tuning fork sensors in atomic force microscopy

Science.gov (United States)

Ebeling, Daniel; Zhong, Qigang; Ahles, Sebastian; Chi, Lifeng; Wegner, Hermann A.; Schirmeisen, André

2017-05-01

We demonstrate the ability of resolving the chemical structure of single organic molecules using non-contact atomic force microscopy with higher normal eigenmodes of quartz tuning fork sensors. In order to achieve submolecular resolution, CO-functionalized tips at low temperatures are used. The tuning fork sensors are operated in ultrahigh vacuum in the frequency modulation mode by exciting either their first or second eigenmode. Despite the high effective spring constant of the second eigenmode (on the order of several tens of kN/m), the force sensitivity is sufficiently high to achieve atomic resolution above the organic molecules. This is observed for two different tuning fork sensors with different tip geometries (small tip vs. large tip). These results represent an important step towards resolving the chemical structure of single molecules with multifrequency atomic force microscopy techniques where two or more eigenmodes are driven simultaneously.

Apoptosis-like yeast cell death in response to DNA damage and replication defects

Energy Technology Data Exchange (ETDEWEB)

Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D' Urso, Gennaro; Huberman, Joel A

2003-11-27

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.

Apoptosis-like yeast cell death in response to DNA damage and replication defects

International Nuclear Information System (INIS)

Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D'Urso, Gennaro; Huberman, Joel A.

2003-01-01

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication

Far-travelled permian chert of the North Fork terrane, Klamath mountains, California

Science.gov (United States)

Mankinen, E.A.; Irwin, W.P.; Blome, C.D.

1996-01-01

Permian chert in the North Fork terrane and correlative rocks of the Klamath Mountains province has a remanent magnetization that is prefolding and presumably primary. Paleomagnetic results indicate that the chert formed at a paleolatitude of 8.6?? ?? 2.5?? but in which hemisphere remains uncertain. This finding requires that these rocks have undergone at least 8.6?? ?? 4.4?? of northward transport relative to Permian North America since their deposition. Paleontological evidence suggests that the Permian limestone of the Eastern Klamath terrane originated thousands of kilometers distant from North America. The limestone of the North Fork terrane may have formed at a similar or even greater distance as suggested by its faunal affinity to the Eastern Klamath terrane and more westerly position. Available evidence indicates that convergence of the North Fork and composite Central Metamorphic-Eastern Klamath terranes occurred during Triassic or Early Jurassic time and that their joining together was a Middle Jurassic event. Primary and secondary magnetizations indicate that the new composite terrane containing these and other rocks of the Western Paleozoic and Triassic belt behaved as a single rigid block that has been latitudinally concordant with the North American craton since Middle Jurassic time.

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

Science.gov (United States)

Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

2015-09-01

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Derek L Lindstrom

2011-03-01

Full Text Available Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array. As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

Integrating Salmon Recovery, Clean Water Act Compliance, Restoration, and Climate Change Impacts in the South Fork Nooksack River

Science.gov (United States)

"The South Fork Nooksack River (SFNR) is an important tributary to the Nooksack River, Bellingham Bay, and the Salish Sea. The South Fork Nooksack River comprises one of the 22 independent populations of spring Chinook in the Puget Sound Chinook Evolutionarily Significant Un...

Turbo Charge CPU Utilization in Fork/Join Using the ManagedBlocker

CERN Multimedia

CERN. Geneva

2017-01-01

Fork/Join is a framework for parallelizing calculations using recursive decomposition, also called divide and conquer. These algorithms occasionally end up duplicating work, especially at the beginning of the run. We can reduce wasted CPU cycles by implementing a reserved caching scheme. Before a task starts its calculation, it tries to reserve an entry in the shared map. If it is successful, it immediately begins. If not, it blocks until the other thread has finished its calculation. Unfortunately this might result in a significant number of blocked threads, decreasing CPU utilization. In this talk we will demonstrate this issue and offer a solution in the form of the ManagedBlocker. Combined with the Fork/Join, it can keep parallelism at the desired level.

Burnup verification measurements at a US nuclear utility using the FORK measurement system

International Nuclear Information System (INIS)

Ewing, R.I.; Bosler, G.E.; Walden, G.

1993-01-01

The FORK measurement system, designed at Los Alamos National Laboratory (LANL) for the International Atomic Energy Agency (IAEA) safeguards program, has been used to examine spent reactor fuel assemblies at Duke Power Company's Oconee Nuclear Station. The FORK system measures the passive neutron and gamma-ray emission from spent fuel assemblies while in the storage pool. These measurements can be correlated with burnup and cooling time, and can be used to verify the reactor site records. Verification measurements may be used to help ensure nuclear criticality safety when burnup credit is applied to spent fuel transport and storage systems. By taking into account the reduced reactivity of spent fuel due to its burnup in the reactor, burnup credit results in more efficient and economic transport and storage. The objectives of these tests are to demonstrate the applicability of the FORK system to verify reactor records and to develop optimal procedures compatible with utility operations. The test program is a cooperative effort supported by Sandia National Laboratories, the Electric Power Research Institute (EPRI), Los Alamos National Laboratory, and the Duke Power Company

Gap Junctions

Science.gov (United States)

Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

2013-01-01

Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012. PMID:23723031

SLX-1 is required for maintaining genomic integrity and promoting meiotic noncrossovers in the Caenorhabditis elegans germline.

Directory of Open Access Journals (Sweden)

Takamune T Saito

2012-08-01

Full Text Available Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644 mutants. SLX-1 shows a HIM-18/SLX-4-dependent endonuclease activity toward replication forks, 5'-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.

InterProScan Result: FS906632 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available FS906632 FS906632_3_ORF2 2C529C8D33AE78A2 PFAM PF07962 Swi3 1e-25 T IPR012923 Replication fork protection...to DNA damage stimulus (GO:0006974)|Biological Process: cell cycle (GO:0007049)|Biological Process: replication fork protection (GO:0048478) ...

The ophiolitic North Fork terrane in the Salmon River region, central Klamath Mountains, California

Science.gov (United States)

Ando, C.J.; Irwin, W.P.; Jones, D.L.; Saleeby, J.B.

1983-01-01

The North Fork terrane is an assemblage of ophiolitic and other oceanic volcanic and sedimentary rocks that has been internally imbricated and folded. The ophiolitic rocks form a north-trending belt through the central part of the region and consist of a disrupted sequence of homogeneous gabbro, diabase, massive to pillowed basalt, and interleaved tectonitic harzburgite. U-Pb zircon age data on a plagiogranite pod from the gabbroic unit indicate that at least this part of the igneous sequence is late Paleozoic in age.The ophiolitic belt is flanked on either side by mafic volcanic and volcaniclastic rocks, limestone, bedded chert, and argillite. Most of the chert is Triassic, including much of Late Triassic age, but chert with uncertain stratigraphic relations at one locality is Permian. The strata flanking the east side of the ophiolitic belt face eastward, and depositional contacts between units are for the most part preserved. The strata on the west side of the ophiolitic belt are more highly disrupted than those on the east side, contain chert-argillite melange, and have unproven stratigraphic relation to either the ophiolitic rocks or the eastern strata.Rocks of the North Fork terrane do not show widespread evidence of penetrative deformation at elevated temperatures, except an early tectonitic fabric in the harzburgite. Slip-fiber foliation in serpentinite, phacoidal foliation in chert and mafic rocks, scaly foliation in argillite, and mesoscopic folds in bedded chert are consistent with an interpretation of large-scale anti-formal folding of the terrane about a north-south hinge found along the ophiolitic belt, but other structural interpretations are tenable. The age of folding of North Fork rocks is constrained by the involvement of Triassic and younger cherts and crosscutting Late Jurassic plutons. Deformation in the North Fork terrane must have spanned a short period of time because the terrane is bounded structurally above and below by Middle or Late

Two mechanisms coordinate replication termination by the Escherichia coli Tus–Ter complex

KAUST Repository

Pandey, Manjula

2015-07-13

The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus–Ter complex. The T7 replisome was arrested at the non-permissive end of Tus–Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus–Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.

A case with concurrent duplication, triplication, and uniparental isodisomy at 1q42.12-qter supporting microhomology-mediated break-induced replication model for replicative rearrangements.

Science.gov (United States)

Kohmoto, Tomohiro; Okamoto, Nana; Naruto, Takuya; Murata, Chie; Ouchi, Yuya; Fujita, Naoko; Inagaki, Hidehito; Satomura, Shigeko; Okamoto, Nobuhiko; Saito, Masako; Masuda, Kiyoshi; Kurahashi, Hiroki; Imoto, Issei

2017-01-01

Complex genomic rearrangements (CGRs) consisting of interstitial triplications in conjunction with uniparental isodisomy (isoUPD) have rarely been reported in patients with multiple congenital anomalies (MCA)/intellectual disability (ID). One-ended DNA break repair coupled with microhomology-mediated break-induced replication (MMBIR) has been recently proposed as a possible mechanism giving rise to interstitial copy number gains and distal isoUPD, although only a few cases providing supportive evidence in human congenital diseases with MCA have been documented. Here, we report on the chromosomal microarray (CMA)-based identification of the first known case with concurrent interstitial duplication at 1q42.12-q42.2 and triplication at 1q42.2-q43 followed by isoUPD for the remainder of chromosome 1q (at 1q43-qter). In distal 1q duplication/triplication overlapping with 1q42.12-q43, variable clinical features have been reported, and our 25-year-old patient with MCA/ID presented with some of these frequently described features. Further analyses including the precise mapping of breakpoint junctions within the CGR in a sequence level suggested that the CGR found in association with isoUPD in our case is a triplication with flanking duplications, characterized as a triplication with a particularly long duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) structure. Because microhomology was observed in both junctions between the triplicated region and the flanking duplicated regions, our case provides supportive evidence for recently proposed replication-based mechanisms, such as MMBIR, underlying the formation of CGRs + isoUPD implicated in chromosomal disorders. To the best of our knowledge, this is the first case of CGRs + isoUPD observed in 1q and having DUP-TRP/INV-DUP structure with a long proximal duplication, which supports MMBIR-based model for genomic rearrangements. Molecular cytogenetic analyses using CMA containing single

Gap junctions and memory: an investigation using a single trial discrimination avoidance task for the neonate chick.

Science.gov (United States)

Verwey, L J; Edwards, T M

2010-02-01

Gap junctions are important to how the brain functions but are relatively under-investigated with respect to their contribution towards behaviour. In the present study a single trial discrimination avoidance task was used to investigate the effect of the gap junction inhibitor 18-alpha-glycyrrhetinic acid (alphaGA) on retention. Past studies within our research group have implied a potential role for gap junctions during the short-term memory (STM) stage which decays by 15 min post-training. A retention function study comparing 10 microM alphaGA and vehicle given immediately post-training demonstrated a significant main effect for drug with retention loss at all times of test (10-180 min post-training). Given that the most common gap junction in the brain is that forming the astrocytic network it is reasonable to conclude that alphaGA was acting upon these. To confirm this finding and interpretation two additional investigations were undertaken using endothelin-1 (ET-1) and ET-1+tolbutamide. Importantly, a retention function study using 10nM ET-1 replicated the retention loss observed for alphaGA. In order to confirm that ET-1 was acting on astrocytic gap junctions the amnestic action of ET-1 was effectively challenged with increasing concentrations of tolbutamide. The present findings suggest that astrocytic gap junctions are important for memory processing. Copyright 2009 Elsevier Inc. All rights reserved.

Flood-inundation maps for the East Fork White River near Bedford, Indiana

Science.gov (United States)

Fowler, Kathleen K.

2014-01-01

Digital flood-inundation maps for an 1.8-mile reach of the East Fork White River near Bedford, Indiana (Ind.) were created by the U.S. Geological Survey (USGS) in cooperation with the Indiana Department of Transportation. The inundation maps, which can be accessed through the USGS Flood Inundation Mapping Science Web site at http://water.usgs.gov/osw/flood_inundation/ depict estimates of the areal extent and depth of flooding corresponding to selectedwater levels (stages) at USGS streamgage 03371500, East Fork White River near Bedford, Ind. Current conditions for estimating near-real-time areas of inundation using USGS streamgage information may be obtained on the Internet at http://waterdata.usgs.gov/in/nwis/uv?site_no=03371500. In addition, information has been provided to the National Weather Service (NWS) for incorporation into their Advanced Hydrologic Prediction Service (AHPS) flood warning system (http://water.weather.gov/ahps/). The NWS forecasts flood hydrographs at many places that are often colocated with USGS streamgages, including the East Fork White River near Bedford, Ind. NWS-forecasted peak-stage information may be used in conjunction with the maps developed in this study to show predicted areas of flood inundation. For this study, flood profiles were computed for the East Fork White River reach by means of a one-dimensional step-backwater model. The hydraulic model was calibrated by using the most current stage-discharge relations at USGS streamgage 03371500, East Fork White River near Bedford, Ind., and documented high-water marks from the flood of June 2008. The calibrated hydraulic model was then used to determine 20 water-surface profiles for flood stages at 1-foot intervals referenced to the streamgage datum and ranging from bankfull to the highest stage of the current stage-discharge rating curve. The simulated water-surface profiles were then combined with a geographic information system (GIS) digital elevation model (DEM, derived from

Four-junction superconducting circuit

Science.gov (United States)

Qiu, Yueyin; Xiong, Wei; He, Xiao-Ling; Li, Tie-Fu; You, J. Q.

2016-01-01

We develop a theory for the quantum circuit consisting of a superconducting loop interrupted by four Josephson junctions and pierced by a magnetic flux (either static or time-dependent). In addition to the similarity with the typical three-junction flux qubit in the double-well regime, we demonstrate the difference of the four-junction circuit from its three-junction analogue, including its advantages over the latter. Moreover, the four-junction circuit in the single-well regime is also investigated. Our theory provides a tool to explore the physical properties of this four-junction superconducting circuit. PMID:27356619

A Transimpedance Amplifier for Remotely Located Quartz Tuning Forks

OpenAIRE

Kleinbaum, Ethan; Csathy, Gabor

2012-01-01

The cable capacitance in cryogenic and high vacuum applications of quartz tuning forks imposes severe constraints on the bandwidth and noise performance of the measurement. We present a single stage low noise transimpedance amplifier with a bandwidth exceeding 1 MHz and provide an in-depth analysis of the dependence of the amplifier parameters on the cable capacitance.

Distance control for a near-field scanning microwave microscope in liquid using a quartz tuning fork

International Nuclear Information System (INIS)

Kim, Song Hul; Yoo, Hyun Jun; Yoo, Hyung Geun; Lee, Kie Jin

2004-01-01

We demonstrate a scanning near-field microwave microscope (NSMM) in the liquid environment using a tuning fork shear-force feedback method to control the distance between tip and sample. The probe tip for the NSMM is only immersed in water and attached to one prong of a quartz tuning fork and directly coupled to a high-quality dielectric resonator at an operating frequency f = 4.5-5.5 GHz. This distance control method is independent of the local microwave characteristics. The amplitude of the tuning fork was used as a set point of the distance control parameter in the liquid. To demonstrate the ability of the distance regulation system, we present the NSMM images of a copper film in air and liquid without and with readjusting the distance set point and a DNA film image in buffer solution.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

Science.gov (United States)

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Implementing farm-to-fork traceability in Tanzania

CSIR Research Space (South Africa)

Van Dyk, FE

2005-08-01

Full Text Available stream_source_info Van Dyk2_2005.pdf.txt stream_content_type text/plain stream_size 10949 Content-Encoding UTF-8 stream_name Van Dyk2_2005.pdf.txt Content-Type text/plain; charset=UTF-8 Copyright @ CSIR 2005 www....csir.co.za Implementing farm-to-fork traceability in Tanzania Esbeth van Dyk CSIR Centre for Logistics ORSSA/SAIIE August 2005 Copyright @ CSIR 2005 www.csir.co.za Structure • Why traceability? • Legislation • Tanzania project • Recordkeeping in coffee...

Ballistic Graphene Josephson Junctions from the Short to the Long Junction Regimes.

Science.gov (United States)

Borzenets, I V; Amet, F; Ke, C T; Draelos, A W; Wei, M T; Seredinski, A; Watanabe, K; Taniguchi, T; Bomze, Y; Yamamoto, M; Tarucha, S; Finkelstein, G

2016-12-02

We investigate the critical current I_{C} of ballistic Josephson junctions made of encapsulated graphene-boron-nitride heterostructures. We observe a crossover from the short to the long junction regimes as the length of the device increases. In long ballistic junctions, I_{C} is found to scale as ∝exp(-k_{B}T/δE). The extracted energies δE are independent of the carrier density and proportional to the level spacing of the ballistic cavity. As T→0 the critical current of a long (or short) junction saturates at a level determined by the product of δE (or Δ) and the number of the junction's transversal modes.

Equivalent Josephson junctions

International Nuclear Information System (INIS)

Boyadzhiev, T.L.; ); Semerdzhieva, E.G.; Shukrinov, Yu.M.; Fiziko-Tekhnicheskij Inst., Dushanbe

2008-01-01

The magnetic field dependences of critical current are numerically constructed for a long Josephson junction with a shunt- or resistor-type microscopic inhomogeneities and compared to the critical curve of a junction with exponentially varying width. The numerical results show that it is possible to replace the distributed inhomogeneity of a long Josephson junction by an inhomogeneity localized at one of its ends, which has certain technological advantages. It is also shown that the critical curves of junctions with exponentially varying width and inhomogeneities localized at the ends are unaffected by the mixed fluxon-antifluxon distributions of the magnetic flux [ru

Dual parasitism of Fork-tailed Drongos by African and Jacobin ...

African Journals Online (AJOL)

Different species of brood parasitic birds, which lay their eggs in the nests of host foster-parents, rarely target the same host species population. We report brood parasitism of Fork-tailed Drongos Dicrurus adsimilis in the southern Kalahari Desert by both African Cuckoo Cuculus gularis and Jacobin Cuckoo Clamator ...

Water-quality trends for selected sampling sites in the upper Clark Fork Basin, Montana, water years 1996-2010

Science.gov (United States)

Sando, Steven K.; Vecchia, Aldo V.; Lorenz, David L.; Barnhart, Elliott P.

2014-01-01

A large-scale trend analysis was done on specific conductance, selected trace elements (arsenic, cadmium, copper, iron, lead, manganese, and zinc), and suspended-sediment data for 22 sites in the upper Clark Fork Basin for water years 1996–2010. Trend analysis was conducted by using two parametric methods: a time-series model (TSM) and multiple linear regression on time, streamflow, and season (MLR). Trend results for 1996–2010 indicate moderate to large decreases in flow-adjusted concentrations (FACs) and loads of copper (and other metallic elements) and suspended sediment in Silver Bow Creek upstream from Warm Springs. Deposition of metallic elements and suspended sediment within Warm Springs Ponds substantially reduces the downstream transport of those constituents. However, mobilization of copper and suspended sediment from floodplain tailings and stream banks in the Clark Fork reach from Galen to Deer Lodge is a large source of metallic elements and suspended sediment, which also affects downstream transport of those constituents. Copper and suspended-sediment loads mobilized from within this reach accounted for about 40 and 20 percent, respectively, of the loads for Clark Fork at Turah Bridge (site 20); whereas, streamflow contributed from within this reach only accounted for about 8 percent of the streamflow at Turah Bridge. Minor changes in FACs and loads of copper and suspended sediment are indicated for this reach during 1996–2010. Clark Fork reaches downstream from Deer Lodge are relatively smaller sources of metallic elements than the reach from Galen to Deer Lodge. In general, small decreases in loads and FACs of copper and suspended sediment are indicated for Clark Fork sites downstream from Deer Lodge during 1996–2010. Thus, although large decreases in FACs and loads of copper and suspended sediment are indicated for Silver Bow Creek upstream from Warm Springs, those large decreases are not translated to the more downstream reaches largely

Junction and circuit fabrication

International Nuclear Information System (INIS)

Jackel, L.D.

1980-01-01

Great strides have been made in Josephson junction fabrication in the four years since the first IC SQUID meeting. Advances in lithography have allowed the production of devices with planar dimensions as small as a few hundred angstroms. Improved technology has provided ultra-high sensitivity SQUIDS, high-efficiency low-noise mixers, and complex integrated circuits. This review highlights some of the new fabrication procedures. The review consists of three parts. Part 1 is a short summary of the requirements on junctions for various applications. Part 2 reviews intergrated circuit fabrication, including tunnel junction logic circuits made at IBM and Bell Labs, and microbridge radiation sources made at SUNY at Stony Brook. Part 3 describes new junction fabrication techniques, the major emphasis of this review. This part includes a discussion of small oxide-barrier tunnel junctions, semiconductor barrier junctions, and microbridge junctions. Part 3 concludes by considering very fine lithography and limitations to miniaturization. (orig.)

Editorial: 3Rs tightly intertwined to maintain genome stability

DEFF Research Database (Denmark)

Lisby, Michael; Mortensen, Uffe H.

2017-01-01

, replication of damaged DNA results in stalled replication forks that await DNA damage repair before replication can be resumed. In turn, the repair of most lesions depends on processes involving DNA synthesis. At the same time, the stalled forks may engage in recombination, either as part of a controlled...... repair process or by accident, just because it can, with the risk of producing genome rearrangements and loss of heterozygosity. The set of reviews presented in this thematic issue (https://academic-oup-com.proxy.findit.dtu.dk/femsyr/pages/replication_recombination_and_repair) of FEMSYR has been selected...

Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication.

Science.gov (United States)

Kjær, Jonas; Belsham, Graham J

2018-04-15

Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed "ribosome skipping" or "StopGo." Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E 14 , S 15 , and N 16 within the 2A sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P 17 , G 18 , or P 19 , which displayed little or no "cleavage" activity in vitro , were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E 14 , S 15 , N 16 , and P 19 resulted in partial "cleavage" of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational "cleavage." Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at

Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.

Science.gov (United States)

Pandey, Manjula; Elshenawy, Mohamed M; Jergic, Slobodan; Takahashi, Masateru; Dixon, Nicholas E; Hamdan, Samir M; Patel, Smita S

2015-07-13

The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus-Ter complex. The T7 replisome was arrested at the non-permissive end of Tus-Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus-Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

U.S. Geological Survey 2013 assessment of undiscovered resources in the Bakken and Three Forks Formations of the U.S. Williston Basin Province

Science.gov (United States)

Gaswirth, Stephanie B.; Marra, Kristen R.

2014-01-01

The Upper Devonian Three Forks and Upper Devonian to Lower Mississippian Bakken Formations comprise a major United States continuous oil resource. Current exploitation of oil is from horizontal drilling and hydraulic fracturing of the Middle Member of the Bakken and upper Three Forks, with ongoing exploration of the lower Three Forks, and the Upper, Lower, and Pronghorn Members of the Bakken Formation. In 2008, the U.S. Geological Survey (USGS) estimated a mean of 3.65 billion bbl of undiscovered, technically recoverable oil resource within the Bakken Formation. The USGS recently reassessed the Bakken Formation, which included an assessment of the underlying Three Forks Formation. The Pronghorn Member of the Bakken Formation, where present, was included as part of the Three Forks assessment due to probable fluid communication between reservoirs. For the Bakken Formation, five continuous and one conventional assessment units (AUs) were defined. These AUs are modified from the 2008 AU boundaries to incorporate expanded geologic and production information. The Three Forks Formation was defined with one continuous and one conventional AU. Within the continuous AUs, optimal regions of hydrocarbon recovery, or “sweet spots,” were delineated and estimated ultimate recoveries were calculated for each continuous AU. Resulting undiscovered, technically recoverable resource estimates were 3.65 billion bbl for the five Bakken continuous oil AUs and 3.73 billion bbl for the Three Forks Continuous Oil AU, generating a total mean resource estimate of 7.38 billion bbl. The two conventional AUs are hypothetical and represent a negligible component of the total estimated resource (8 million barrels of oil).

Macroscopic quantum tunneling in Josephson tunnel junctions and Coulomb blockade in single small tunnel junctions

International Nuclear Information System (INIS)

Cleland, A.N.

1991-04-01

Experiments investigating the process of macroscopic quantum tunneling in a moderately-damped, resistively shunted, Josephson junction are described, followed by a discussion of experiments performed on very small capacitance normal-metal tunnel junctions. The experiments on the resistively-shunted Josephson junction were designed to investigate a quantum process, that of the tunneling of the Josephson phase variable under a potential barrier, in a system in which dissipation plays a major role in the dynamics of motion. All the parameters of the junction were measured using the classical phenomena of thermal activation and resonant activation. Theoretical predictions are compared with the experimental results, showing good agreement with no adjustable parameters; the tunneling rate in the moderately damped (Q ∼ 1) junction is seen to be reduced by a factor of 300 from that predicted for an undamped junction. The phase is seen to be a good quantum-mechanical variable. The experiments on small capacitance tunnel junctions extend the measurements on the larger-area Josephson junctions from the region in which the phase variable has a fairly well-defined value, i.e. its wavefunction has a narrow width, to the region where its value is almost completely unknown. The charge on the junction becomes well-defined and is predicted to quantize the current through the junction, giving rise to the Coulomb blockade at low bias. I present the first clear observation of the Coulomb blockade in single junctions. The electrical environment of the tunnel junction, however, strongly affects the behavior of the junction: higher resistance leads are observed to greatly sharpen the Coulomb blockade over that seen with lower resistance leads. I present theoretical descriptions of how the environment influences the junctions; comparisons with the experimental results are in reasonable agreement

The ammonoids from the Three Forks Shale (Late Devonian of Montana

Directory of Open Access Journals (Sweden)

D. Korn

2006-08-01

Full Text Available The ammonoid fauna from the Late Devonian Three Forks Shale of Montana is revised. Six taxa were recognised, which belong to the genera Tornoceras, Pernoceras, Raymondiceras, Platyclymenia, Pleuroclymenia, and Carinoclymenia. The ammonoid assemblage suggests a stratigraphic position within the middle Famennian, most probably the Platyclymenia annulata Zone. The ammonoids display extreme septal crowding in intermediate as well as adult growth stages, which can be regarded as evidence for instable palaeoecological conditions during lifetime of the animals. Die Ammonoideenfauna aus dem oberdevonischen Three Forks Shale von Montana wird revidiert. Sechs Taxa werden unterschieden; sie gehören zu den Gattungen Tornoceras, Pernoceras, Raymondiceras, Platyclymenia, Pleuroclymenia und Carinoclymenia. Die Ammonoideen-Vergesellschaftung spricht für eine stratigraphische Position im mittleren Famennium, wahrscheinlich in der Platyclymenia annulata Zone. Die Ammonoideen zeigen auffällige Drängung der Septen in intermediären und adulten Wachstumsstadien, die als Hinweis auf instabile Lebensbedingungen für die Tiere gewertet werden kann. doi:10.1002/mmng.200600008

Note: a transimpedance amplifier for remotely located quartz tuning forks.

Science.gov (United States)

Kleinbaum, Ethan; Csáthy, Gábor A

2012-12-01

The cable capacitance in cryogenic and high vacuum applications of quartz tuning forks imposes severe constraints on the bandwidth and noise performance of the measurement. We present a single stage low noise transimpedance amplifier with a bandwidth exceeding 1 MHz and provide an in-depth analysis of the dependence of the amplifier parameters on the cable capacitance.

Mean rate of DNA replication and replicon size in the shoot apex of Silence coeli-rosa. During the initial 120 minutes of the first day of floral induction

International Nuclear Information System (INIS)

Ormrod, J.C.; Francis, D.

1986-01-01

28-day-old plants of Silence coeli-rosa were exposed, at 1700 hours, to long day (LD) conditions comprising light of low fluence rate provided by tungsten bulbs, or maintained in darkness as short day (SD) controls. All plants were exposed at 1700 hours to tritiated-(methyl- 3 H)-thymidine for 30, 45, 60, 90, or 120 minutes. Apical domes were isolated and prepared as fiber autoradiographs from which replicon size and rates of DNA replication, per single replication fork were recorded. In SD, replicon size was between 15-20 μm and exposure to LD conditions altered neither replicon size nor the pattern of deployment of replicons during S-phase relative to the SD controls. However, the mean rate of replication in LD was 8.7 μm h -1 compared with 5.2 μm h -1 in SD. Thus, exposure to LD resulted in a 1.7-fold increase in the rate of DNA replication relative to the SD controls. This rapid increase in replication rate, detectable within 30 minutes of the start of the LD is discussed in relation to changes known to occur to the cell cycle in Silene during the first day of floral induction. (Author)

Pitch Fork: A Novel tactile Digital Musical Instrument

OpenAIRE

Williams, Peter; Overholt, Daniel

2017-01-01

Pitch Fork is a prototype of an alternate, actuated digital musical instrument (DMI). It uses 5 infra-red and 4 piezoelectric sensors to control an additive synthesis engine. Iron bars are used as the physical point of contact in interaction with the aim of using material computation to control aspects of the digitally produced sound. This choice of material was also chosen to affect player experience. Sensor readings are relayed to a Macbook via an Arduino Mega. Mappings and audio output sig...

Habitat-dependent interactions between two size-classes of juvenile steelhead in a small stream

Science.gov (United States)

Bret C. Harvey; Rodney J. Nakamoto

1997-01-01

Abstract - The presence of small steelhead (Oncorhynchus mykiss; averaging 55 mm fork length) influenced the growth of larger juvenile steelhead (90 mm fork length) during a 6-week experiment conducted in North Fork Caspar Creek, California, in summer 1994. In fenced replicate deep stream sections in this small stream, growth of the larger steelhead was greater in...

Josephson junction arrays

International Nuclear Information System (INIS)

Bindslev Hansen, J.; Lindelof, P.E.

1985-01-01

In this review we intend to cover recent work involving arrays of Josephson junctions. The work on such arrays falls naturally into three main areas of interest: 1. Technical applications of Josephson junction arrays for high-frequency devices. 2. Experimental studies of 2-D model systems (Kosterlitz-Thouless phase transition, commensurate-incommensurate transition in frustrated (flux) lattices). 3. Investigations of phenomena associated with non-equilibrium superconductivity in and around Josephson junctions (with high current density). (orig./BUD)

Qualitative Assessment: Evaluating the Impacts of Climate Change on Endangered Species Act Recovery Actions for the South Fork Nooksack River, WA

Science.gov (United States)

The South Fork Nooksack River (South Fork) is located in northwest Washington State and is home to nine species of Pacific salmon, including Nooksack early Chinook (aka, spring Chinook salmon), an iconic species for the Nooksack Indian Tribe. The quantity of salmon in the South F...

Electronic thermometry in tunable tunnel junction

Science.gov (United States)

Maksymovych, Petro

2016-03-15

A tunable tunnel junction thermometry circuit includes a variable width tunnel junction between a test object and a probe. The junction width is varied and a change in thermovoltage across the junction with respect to the change in distance across the junction is determined. Also, a change in biased current with respect to a change in distance across the junction is determined. A temperature gradient across the junction is determined based on a mathematical relationship between the temperature gradient, the change in thermovoltage with respect to distance and the change in biased current with respect to distance. Thermovoltage may be measured by nullifying a thermoelectric tunneling current with an applied voltage supply level. A piezoelectric actuator may modulate the probe, and thus the junction width, to vary thermovoltage and biased current across the junction. Lock-in amplifiers measure the derivatives of the thermovoltage and biased current modulated by varying junction width.

Dynamics of Josephson junction arrays

International Nuclear Information System (INIS)

Hadley, P.

1989-01-01

The dynamics of Josephson junction arrays is a topic that lies at the intersection of the fields of nonlinear dynamics and Josephson junction technology. The series arrays considered here consist of several rapidly oscillating Josephson junctions where each junction is coupled equally to every other junction. The purpose of this study is to understand phaselocking and other cooperative dynamics of this system. Previously, little was known about high dimensional nonlinear systems of this sort. Numerical simulations are used to study the dynamics of these arrays. Three distinct types of periodic solutions to the array equations were observed as well as period doubled and chaotic solutions. One of the periodic solutions is the symmetric, in-phase solution where all of the junctions oscillate identically. The other two periodic solutions are symmetry-broken solutions where all of the junction do not oscillate identically. The symmetry-broken solutions are highly degenerate. As many as (N - 1) stable solutions can coexist for an array of N junctions. Understanding the stability of these several solutions and the transitions among them is vital to the design of useful devices

Fork-join and data-driven execution models on multi-core architectures: Case study of the FMM

KAUST Repository

Amer, Abdelhalim

2013-01-01

Extracting maximum performance of multi-core architectures is a difficult task primarily due to bandwidth limitations of the memory subsystem and its complex hierarchy. In this work, we study the implications of fork-join and data-driven execution models on this type of architecture at the level of task parallelism. For this purpose, we use a highly optimized fork-join based implementation of the FMM and extend it to a data-driven implementation using a distributed task scheduling approach. This study exposes some limitations of the conventional fork-join implementation in terms of synchronization overheads. We find that these are not negligible and their elimination by the data-driven method, with a careful data locality strategy, was beneficial. Experimental evaluation of both methods on state-of-the-art multi-socket multi-core architectures showed up to 22% speed-ups of the data-driven approach compared to the original method. We demonstrate that a data-driven execution of FMM not only improves performance by avoiding global synchronization overheads but also reduces the memory-bandwidth pressure caused by memory-intensive computations. © 2013 Springer-Verlag.

Hydrogeology and simulated groundwater flow and availability in the North Fork Red River aquifer, southwest Oklahoma, 1980–2013

Science.gov (United States)

Smith, S. Jerrod; Ellis, John H.; Wagner, Derrick L.; Peterson, Steven M.

2017-09-28

On September 8, 1981, the Oklahoma Water Resources Board established regulatory limits on the maximum annual yield of groundwater (343,042 acre-feet per year) and equal-proportionate-share (EPS) pumping rate (1.0 acre-foot per acre per year) for the North Fork Red River aquifer. The maximum annual yield and EPS were based on a hydrologic investigation that used a numerical groundwater-flow model to evaluate the effects of potential groundwater withdrawals on groundwater availability in the North Fork Red River aquifer. The Oklahoma Water Resources Board is statutorily required (every 20 years) to update the hydrologic investigation on which the maximum annual yield and EPS were based. Because 20 years have elapsed since the final order was issued, the U.S. Geological Survey, in cooperation with the Oklahoma Water Resources Board, conducted an updated hydrologic investigation and evaluated the effects of potential groundwater withdrawals on groundwater flow and availability in the North Fork Red River aquifer in Oklahoma. This report describes a hydrologic investigation of the North Fork Red River aquifer that includes an updated summary of the aquifer hydrogeology. As part of this investigation, groundwater flow and availability were simulated by using a numerical groundwater-flow model.The North Fork Red River aquifer in Beckham, Greer, Jackson, Kiowa, and Roger Mills Counties in Oklahoma is composed of about 777 square miles (497,582 acres) of alluvium and terrace deposits along the North Fork Red River and tributaries, including Sweetwater Creek, Elk Creek, Otter Creek, and Elm Fork Red River. The North Fork Red River is the primary source of surface-water inflow to Lake Altus, which overlies the North Fork Red River aquifer. Lake Altus is a U.S. Bureau of Reclamation reservoir with the primary purpose of supplying irrigation water to the Lugert-Altus Irrigation District.A hydrogeologic framework was developed for the North Fork Red River aquifer and included a

Dynamic behavior of tuning fork shear-force structures in a SNOM system

Energy Technology Data Exchange (ETDEWEB)

Gao, Fengli [Department of Engineering Mechanics, AML, CNMM, Tsinghua University, Beijing 100084 (China); Li, Xide, E-mail: lixide@tsinghua.edu.cn [Department of Engineering Mechanics, AML, CNMM, Tsinghua University, Beijing 100084 (China); Wang, Jia [State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instruments, Tsinghua University, Beijing 100084 (China); Fu, Yu [Temasek Laboratories, Nanyang Technological University, 50 Nanyang Drive, 637553 (Singapore)

2014-07-01

Piezoelectric tuning fork shear-force structures are widely used as a distance control unit in a scanning near-field optical microscopy. However, the complex dynamic behavior among the micro-tuning forks (TFs), optical fiber probes, and the probe–surface interactions is still a crucial issue to achieve high-resolution imaging or near-field interaction inspections. Based on nonlinear beam tension-bending vibration theory, vibration equations in both longitudinal and lateral directions have been established when the TF structure and the optical fiber are treated as deformable structures. The relationship of the probe–surface interaction induced by Van der Waals force has been analyzed and the corresponding numerical results used to describe the vibrational behavior of the probe approaching the sample surface are obtained. Meanwhile, the viscous resistance of the liquid film on the sample surface has also been investigated using linear beam-bending vibration theory. Experiments testing the interaction between the probe and the water film on a single crystal silicon wafer have been carried out and the viscous resistance of the water film was estimated using the established equations. Finally, to use the TF-probe structure as a force sensor, the relation between the dynamic response of the TF-probe system and an external force on the probe tip was obtained. - Highlights: • Nonlinear vibration equation is established for a deformable tuning fork probe assembly. • Probe–sample interactions induced by Van der Waals force and viscous resistance are investigated. • The viscous resistance between the probe and the water film is estimated using testing results.

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

Directory of Open Access Journals (Sweden)

Greicy H Goto

2015-08-01

Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

The Model Analysis of a Complex Tuning Fork Probe and Its Application in Bimodal Atomic Force Microscopy

Directory of Open Access Journals (Sweden)

Zhichao Wu

2017-01-01

Full Text Available A new electromechanical coupling model was built to quantitatively analyze the tuning fork probes, especially the complex ones. A special feature of a novel, soft tuning fork probe, that the second eigenfrequency of the probe was insensitive to the effective force gradient, was found and used in a homemade bimodal atomic force microscopy to measure power dissipation quantitatively. By transforming the mechanical parameters to the electrical parameters, a monotonous and concise method without using phase to calculate the power dissipation was proposed.

Relation fonctionnelle entre le pool de nucléotides et PARP-1 : une nouvelle source d'instabilité génétique

OpenAIRE

Gemble , Simon

2015-01-01

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, w...

Tight junctions and human diseases.

Science.gov (United States)

Sawada, Norimasa; Murata, Masaki; Kikuchi, Keisuke; Osanai, Makoto; Tobioka, Hirotoshi; Kojima, Takashi; Chiba, Hideki

2003-09-01

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

Science.gov (United States)

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter. Copyright © 2017 Wang et al.

A single-gradient junction technique to replace multiple-junction shifts for craniospinal irradiation treatment

International Nuclear Information System (INIS)

Hadley, Austin; Ding, George X.

2014-01-01

Craniospinal irradiation (CSI) requires abutting fields at the cervical spine. Junction shifts are conventionally used to prevent setup error–induced overdosage/underdosage from occurring at the same location. This study compared the dosimetric differences at the cranial-spinal junction between a single-gradient junction technique and conventional multiple-junction shifts and evaluated the effect of setup errors on the dose distributions between both techniques for a treatment course and single fraction. Conventionally, 2 lateral brain fields and a posterior spine field(s) are used for CSI with weekly 1-cm junction shifts. We retrospectively replanned 4 CSI patients using a single-gradient junction between the lateral brain fields and the posterior spine field. The fields were extended to allow a minimum 3-cm field overlap. The dose gradient at the junction was achieved using dose painting and intensity-modulated radiation therapy planning. The effect of positioning setup errors on the dose distributions for both techniques was simulated by applying shifts of ± 3 and 5 mm. The resulting cervical spine doses across the field junction for both techniques were calculated and compared. Dose profiles were obtained for both a single fraction and entire treatment course to include the effects of the conventional weekly junction shifts. Compared with the conventional technique, the gradient-dose technique resulted in higher dose uniformity and conformity to the target volumes, lower organ at risk (OAR) mean and maximum doses, and diminished hot spots from systematic positioning errors over the course of treatment. Single-fraction hot and cold spots were improved for the gradient-dose technique. The single-gradient junction technique provides improved conformity, dose uniformity, diminished hot spots, lower OAR mean and maximum dose, and one plan for the entire treatment course, which reduces the potential human error associated with conventional 4-shifted plans

Flood disaster preparedness: a retrospect from Grand Forks, North Dakota.

Science.gov (United States)

Siders, C; Jacobson, R

1998-01-01

Natural disasters often come without warning. The clinical, financial, and business risks can be enormous. Grand Forks' (ND) healthcare systems experienced a flooding disaster of unprecedented proportions in April of 1997. Planned and practiced disaster and evacuation procedures can significantly reduce a healthcare facilities' risk to life, health, and safety. This article retrospectively analyzes disaster preparation and the complete evacuation of the facilities' patients.

Macroscopic quantum tunneling in Josephson tunnel junctions and Coulomb blockade in single small tunnel junctions

International Nuclear Information System (INIS)

Cleland, A.N.

1991-01-01

Experiments investigated the process of macroscopic quantum tunneling in a moderately-damped, resistively shunted, Josephson junction are described, followed by a discussion of experiments performed on very-small-capacitance normal-metal tunnel junctions. The experiments on the resistively-shunted Josephson junction were designed to investigate a quantum process, that of the tunneling of the Josephson-phase variable under a potential barrier, in a system in which dissipation plays a major role in the dynamics of motion. All the parameters of the junction were measured using the classical phenomena of thermal activation and resonant activation. Theoretical predictions are compared with the experimental results, showing good agreement with no adjustable parameters. The experiments on small-capacitance tunnel junctions extend the measurements on the large-area Josephson junctions from the region in which the phase variable has a fairly well-defined value, i.e. its wave function has a narrow width, to the region where its value is almost completely unknown. The charge on the junction becomes well-defined and is predicted to quantize the current through the junction, giving rise to the Coulomb blockade at low bias

Increasing gap junctional coupling: a tool for dissecting the role of gap junctions

DEFF Research Database (Denmark)

Axelsen, Lene Nygaard; Haugan, Ketil; Stahlhut, Martin

2007-01-01

Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes....... In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing...... the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs...

Junction detection and pathway selection

Science.gov (United States)

Peck, Alex N.; Lim, Willie Y.; Breul, Harry T.

1992-02-01

The ability to detect junctions and make choices among the possible pathways is important for autonomous navigation. In our script-based navigation approach where a journey is specified as a script of high-level instructions, actions are frequently referenced to junctions, e.g., `turn left at the intersection.' In order for the robot to carry out these kind of instructions, it must be able (1) to detect an intersection (i.e., an intersection of pathways), (2) know that there are several possible pathways it can take, and (3) pick the pathway consistent with the high level instruction. In this paper we describe our implementation of the ability to detect junctions in an indoor environment, such as corners, T-junctions and intersections, using sonar. Our approach uses a combination of partial scan of the local environment and recognition of sonar signatures of certain features of the junctions. In the case where the environment is known, we use additional sensor information (such as compass bearings) to help recognize the specific junction. In general, once a junction is detected and its type known, the number of possible pathways can be deduced and the correct pathway selected. Then the appropriate behavior for negotiating the junction is activated.

South Fork Salmon River Watershed Restoration, 2008-2009 Annual Report.

Energy Technology Data Exchange (ETDEWEB)

Reaney, Mark D. [Nez Perce Tribe Department of Fisheries Resource Management

2009-04-15

The watershed restoration work elements within the project area, the South Fork Salmon River Watershed, follow the watershed restoration approach adopted by the Nez Perce Tribe Department of Fisheries Resource Management (DFRM) - Watershed Division. The vision of the Nez Perce Tribe DFRM-Watershed Division focuses on protecting, restoring, and enhancing watersheds and treaty resources within the ceded territory of the Nez Perce Tribe under the Treaty of 1855 with the United States Federal Government. The program uses a holistic approach, which encompasses entire watersheds, ridge top to ridge top, emphasizing all cultural aspects and strategies that rely on natural fish production and healthy river ecosystems. The Nez Perce Tribe DFRM-Watershed Division strives towards maximizing historic ecosystem productivity and health for the restoration of anadromous and resident fish populations and the habitat on which all depend on for future generations Originally, this project was funded to create a step/pool stream channel that was appropriate to restore fish passage where the 'Glory Hole Cascade' is currently located at the Stibnite Mine. Due to unforeseen circumstances at the time, the project is unable to move forward as planned and a request for a change in scope of the project and an expansion of the geographic area in which to complete project work was submitted. No additional funds were being requested. The ultimate goal of this project is to work with the holistic, ridge top to ridge top approach to protect and restore the ecological and biological functions of the South Fork Salmon River Watershed to assist in the recovery of threatened and endangered anadromous and resident fish species. FY 2008 Work Elements included two aquatic organism passage (AOP) projects to restore habitat connectivity to two fish-bearing tributaries to the East Fork South Fork Salmon River, Salt and Profile Creeks. The Work Elements also included road survey and assessment

Supramolecular tunneling junctions

NARCIS (Netherlands)

Wimbush, K.S.

2012-01-01

In this study a variety of supramolecular tunneling junctions were created. The basis of these junctions was a self-assembled monolayer of heptathioether functionalized ß-cyclodextrin (ßCD) formed on an ultra-flat Au surface, i.e., the bottom electrode. This gave a well-defined hexagonally packed

Phase diagrams of particles with dissimilar patches: X-junctions and Y-junctions

International Nuclear Information System (INIS)

Tavares, J M; Teixeira, P I C

2012-01-01

We use Wertheim’s first-order perturbation theory to investigate the phase behaviour and the structure of coexisting fluid phases for a model of patchy particles with dissimilar patches (two patches of type A and f B patches of type B). A patch of type α = {A,B} can bond to a patch of type β = {A,B} in a volume v αβ , thereby decreasing the internal energy by ε αβ . We analyse the range of model parameters where AB bonds, or Y-junctions, are energetically disfavoured (ε AB AA /2) but entropically favoured (v AB ≫ v αα ), and BB bonds, or X-junctions, are energetically favoured (ε BB > 0). We show that, for low values of ε BB /ε AA , the phase diagram has three different regions: (i) close to the critical temperature a low-density liquid composed of long chains and rich in Y-junctions coexists with a vapour of chains; (ii) at intermediate temperatures there is coexistence between a vapour of short chains and a liquid of very long chains with X- and Y-junctions; (iii) at low temperatures an ideal gas coexists with a high-density liquid with all possible AA and BB bonds formed. It is also shown that in region (i) the liquid binodal is reentrant (its density decreases with decreasing temperature) for the lower values of ε BB /ε AA . The existence of these three regions is a consequence of the competition between the formation of X- and Y-junctions: X-junctions are energetically favoured and thus dominate at low temperatures, whereas Y-junctions are entropically favoured and dominate at higher temperatures. (paper)

HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

Directory of Open Access Journals (Sweden)

Irna Sufiawati

Full Text Available Herpes simplex virus (HSV types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD. Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.

A vibrating quartz fork - a tool for cryogenic helium research

Czech Academy of Sciences Publication Activity Database

Blažková, Michaela; Člověčko, M.; Eltsov, V. B.; Gažo, E.; de Graaf, R.; Hosio, J.J.; Krusius, M.; Schmoranzer, D.; Schoepe, W.; Skrbek, Ladislav; Skyba, P.; Solntsev, R.E.; Vinen, W. F.

2008-01-01

Roč. 150, - (2008), s. 525-535 ISSN 0022-2291 R&D Projects: GA ČR GA202/05/0218 Grant - others:GAUK(CZ) 7953/2007; Transnational Access Programme(XE) RITA -CT-2003-505313 Institutional research plan: CEZ:AV0Z10100520 Keywords : normal 3He * superfluid 3He * superfluid 4He * turbulence, * cavitation * quartz tuning fork Subject RIV: BK - Fluid Dynamics Impact factor: 1.034, year: 2008

Mercury Content of Sediments in East Fork Poplar Creek: Current Assessment and Past Trends

Energy Technology Data Exchange (ETDEWEB)

Brooks, Scott C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Eller, Virginia A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Dickson, Johnbull O. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Earles, Jennifer E. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Lowe, Kenneth Alan [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Mehlhorn, Tonia L. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Olsen, Todd A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); DeRolph, Christopher R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Watson, David J. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Phillips, Debra H. [Queen' s Univ., Belfast (United Kingdom); Peterson, Mark J. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

2017-01-01

This study provided new information on sediment mercury (Hg) and monomethylmercury (MMHg) content and chemistry. The current inventory of Hg in East Fork Poplar Creek (EFPC) bed sediments was estimated to be 334 kg, which represents a ~67% decrease relative to the initial investigations in 1984. MMHg sediment inventory was estimated to be 44.1 g, lower but roughly similar to past estimates. The results support the relevance and potential impacts of other active and planned investigations within the Mercury Remediation Technology Development for Lower East Fork Poplar Creek project (e.g., assessment and control of bank soil inputs, sorbents for Hg and MMHg removal, re-introduction of freshwater clams to EFPC), and identify gaps in current understanding that represent opportunities to understand controlling variables that may inform future technology development studies.

Common features of a vortex structure in long exponentially shaped Josephson junctions and Josephson junctions with inhomogeneities

International Nuclear Information System (INIS)

Boyadjiev, T.L.; Semerdjieva, E.G.; Shukrinov, Yu.M.

2007-01-01

We study the vortex structure in three different models of the long Josephson junction: the exponentially shaped Josephson junction and the Josephson junctions with the resistor and the shunt inhomogeneities in the barrier layer. For these three models the critical curves 'critical current-magnetic field' are numerically constructed. We develop the idea of the equivalence of the exponentially shaped Josephson junction and the rectangular junction with the distributed inhomogeneity and demonstrate that at some parameters of the shunt and the resistor inhomogeneities in the ends of the junction the corresponding critical curves are very close to the exponentially shaped one

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4α-induced epithelial polarization

International Nuclear Information System (INIS)

Satohisa, Seiro; Chiba, Hideki; Osanai, Makoto; Ohno, Shigeo; Kojima, Takashi; Saito, Tsuyoshi; Sawada, Norimasa

2005-01-01

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4α [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4α triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4α-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4α led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4α provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization

Flexible 2D layered material junctions

Science.gov (United States)

Balabai, R.; Solomenko, A.

2018-03-01

Within the framework of the methods of the electron density functional and the ab initio pseudopotential, we have obtained the valence electron density spatial distribution, the densities of electron states, the widths of band gaps, the charges on combined regions, and the Coulomb potentials for graphene-based flexible 2D layered junctions, using author program complex. It is determined that the bending of the 2D layered junctions on the angle α leads to changes in the electronic properties of these junctions. In the graphene/graphane junction, there is clear charge redistribution with different signs in the regions of junctions. The presence in the heterojunctions of charge regions with different signs leads to the formation of potential barriers. The greatest potential jump is in the graphene/fluorographene junction. The greatest value of the band gap width is in the graphene/graphane junction.

Integrated Outcrop and Subsurface Studies of the Interwell Environment of Carbonate Reservoirs: Clear Fork (Leonaradian Age) Reservoirs, West Texas and New Mexico

International Nuclear Information System (INIS)

Lucia, F.J.; Ruppel, S.C.

2001-01-01

Characterization of cycle and facies architecture on lower Clear Fork and lowermost upper Clear Fork equivalent outcrops in Apache Canyon of Sierra Diablo was complete. The focus of detailed study in Apache Canyon has been the upper Clear Fork section because this interval contains the productive interval in South Wasson field, the preliminary subsurface study area. Parts of three high-frequency sequences (HFS), each 60 to 100 ft thick, are present on the south wall of Apache Canyon. HFS's display an upper-deepening or backstepping pattern associated with longer-term sea level rise. Each HFS is composed of upward-shallowing cycles whose thickness, facies composition, and continuity vary within and between HFS's

Mass loading of selected major and trace elements in Lake Fork Creek near Leadville, Colorado, September-October 2001

Science.gov (United States)

Walton-Day, Katherine; Flynn, Jennifer L.; Kimball, Briant A.; Runkel, Robert L.

2005-01-01

A mass-loading study of Lake Fork Creek of the Arkansas River between Sugarloaf Dam and the mouth was completed in September-October 2001 to help ascertain the following: (1) variation of pH and aqueous constituent concentrations (calcium, sulfate, alkalinity, aluminum, cadmium, copper, iron, manganese, lead, and zinc) and their relation to toxicity standards along the study reach; (2) location and magnitude of sources of metal loading to Lake Fork Creek; (3) amount and locations of metal attenuation; (4) the effect of streamside wetlands on metal transport from contributing mine tunnels; and (5) the effect of organic-rich inflow from the Leadville National Fish Hatchery on water quality in Lake Fork Creek. The study was done in cooperation with the Bureau of Land Management, U.S. Department of Agriculture Forest Service, and U.S. Fish and Wildlife Service. Constituent concentrations and pH showed variable patterns over the study reach. Hardness-based acute and chronic toxicity standards were exceeded for some inflows and some constituents. However, stream concentrations did not exceed standards except for zinc starting in the upper parts of the study reach and extending to just downstream from the inflow from the Leadville National Fish Hatchery. Dilution from that inflow lowered stream zinc concentrations to less than acute and chronic toxicity standards. The uppermost 800 meters of the study reach that contained inflow from the Bartlett, Dinero, and Nelson mine tunnels and the Dinero wetland was the greatest source of loading for manganese and zinc. A middle section of the study reach that extended approximately 2 kilometers upstream from the National Fish Hatchery inflow to just downstream from that inflow was the largest source of aluminum, copper, iron, and lead loading. The loading was partially from the National Fish Hatchery inflow but also from unknown sources upstream from that inflow, possibly ground water. The largest sources for calcium and sulfate

Burnup measurements with the Los Alamos fork detector

International Nuclear Information System (INIS)

Bosler, G.E.; Rinard, P.M.

1991-01-01

The fork detector system can determine the burnup of spent-fuel assemblies. It is a transportable instrument that can be mounted permanently in a spent-fuel pond near a loading area for shipping casks, or be attached to the storage pond bridge for measurements on partially raised spent-fuel assemblies. The accuracy of the predicted burnup has been demonstrated to be as good as 2% from measurements on assemblies in the United States and other countries. Instruments have also been developed at other facilities throughout the world using the same or different techniques, but with similar accuracies. 14 refs., 2 figs., 2 tabs

The Dissolution of Double Holliday Junctions

DEFF Research Database (Denmark)

Bizard, Anna H; Hickson, Ian D

2014-01-01

as "double Holliday junction dissolution." This reaction requires the cooperative action of a so-called "dissolvasome" comprising a Holliday junction branch migration enzyme (Sgs1/BLM RecQ helicase) and a type IA topoisomerase (Top3/TopoIIIα) in complex with its OB (oligonucleotide/oligosaccharide binding......Double Holliday junctions (dHJS) are important intermediates of homologous recombination. The separate junctions can each be cleaved by DNA structure-selective endonucleases known as Holliday junction resolvases. Alternatively, double Holliday junctions can be processed by a reaction known......) fold containing accessory factor (Rmi1). This review details our current knowledge of the dissolution process and the players involved in catalyzing this mechanistically complex means of completing homologous recombination reactions....

The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

Energy Technology Data Exchange (ETDEWEB)

Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.; Perry, J. Jefferson P.; Finger, L. David; Ng, Cliff; Tsai, Miaw-Sheue; Yannone, Steven M.; Tainer, John A.; Campisi, Judith; Cooper, Priscilla K.

2011-04-20

XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

Quantum Junction Solar Cells

KAUST Repository

Tang, Jiang

2012-09-12

Colloidal quantum dot solids combine convenient solution-processing with quantum size effect tuning, offering avenues to high-efficiency multijunction cells based on a single materials synthesis and processing platform. The highest-performing colloidal quantum dot rectifying devices reported to date have relied on a junction between a quantum-tuned absorber and a bulk material (e.g., TiO 2); however, quantum tuning of the absorber then requires complete redesign of the bulk acceptor, compromising the benefits of facile quantum tuning. Here we report rectifying junctions constructed entirely using inherently band-aligned quantum-tuned materials. Realizing these quantum junction diodes relied upon the creation of an n-type quantum dot solid having a clean bandgap. We combine stable, chemically compatible, high-performance n-type and p-type materials to create the first quantum junction solar cells. We present a family of photovoltaic devices having widely tuned bandgaps of 0.6-1.6 eV that excel where conventional quantum-to-bulk devices fail to perform. Devices having optimal single-junction bandgaps exhibit certified AM1.5 solar power conversion efficiencies of 5.4%. Control over doping in quantum solids, and the successful integration of these materials to form stable quantum junctions, offers a powerful new degree of freedom to colloidal quantum dot optoelectronics. © 2012 American Chemical Society.

Distributional Replication

OpenAIRE

Beare, Brendan K.

2009-01-01

Suppose that X and Y are random variables. We define a replicating function to be a function f such that f(X) and Y have the same distribution. In general, the set of replicating functions for a given pair of random variables may be infinite. Suppose we have some objective function, or cost function, defined over the set of replicating functions, and we seek to estimate the replicating function with the lowest cost. We develop an approach to estimating the cheapest replicating function that i...

hSSB1 associates with and promotes stability of the BLM helicase

OpenAIRE

O'BYRNE, KEN

2017-01-01

Background Maintenance of genome stability is critical in human cells. Mutations in or loss of genome stability pathways can lead to a number of pathologies including cancer. hSSB1 is a critical DNA repair protein functioning in the repair and signalling of stalled DNA replication forks, double strand DNA breaks and oxidised DNA lesions. The BLM helicase is central to the repair of both collapsed DNA replication forks and double strand DNA breaks by homologous recombination. Results In this s...

Dynamic force microscopy with quartz tuning forks at high oscillation amplitudes

International Nuclear Information System (INIS)

Labardi, M

2007-01-01

Dynamic force microscopy (DFM) with the self-oscillator (SO) method allows reasonably high scanning rates even with high Q-factors of the resonant force sensor, typical of cantilevers in ultra-high vacuum and of quartz tuning forks. However, due to simpler interpretation of force spectroscopy measurements, small oscillation amplitudes (sub-nm level) are generally preferred. In applications like 'apertureless' scanning near-field optical microscopy (SNOM), oscillation amplitudes of the order of 5-10 nm are needed to increase optical sensitivity and to apply standard optical artefact suppression methods. This motivates the study of the behaviour of tuning forks driven at such high amplitudes, as compared to usual air-operated cantilevers. Both constant-excitation-amplitude (CE) and constant-oscillation-amplitude (CA) modes of SO-DFM are analysed, since the CA mode is more convenient for SNOM applications, denoting remarkable differences. In particular, possible instability effects, previously found in CE mode, are not anticipated for CA mode. It is shown how resonance and approach ('isophase') curves in both modes can be conveniently described in terms of the usual 'normalized frequency shift' γ and of a 'normalized gain' η, defined as a measurement of surface dissipation

Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level

KAUST Repository

Shirbini, Afnan

2017-05-01

Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.

Stream bank and sediment movement associated with 2008 flooding, South Fork Iowa River

Science.gov (United States)

Stream bank erosion can cause substantial damage to riparian systems and impact the use of water downstream. Risks of bank erosion increase during extreme flood events, and frequencies of extreme events may be increasing under changing climate. We assessed bank erosion within the South Fork Iowa Riv...

Correlates of mercury in fish from lakes near Clyde Forks, Ontario, Canada

International Nuclear Information System (INIS)

Ethier, A.L.M.; Scheuhammer, A.M.; Bond, D.E.

2008-01-01

Subsurface soils near Clyde Forks, Ontario, Canada, can have naturally high concentrations of mercury (Hg) from local geological sources. To investigate Hg in local aquatic food webs, Hg was measured in fish dorsal muscle (mainly yellow perch [YP] and pumpkinseed sunfish [PS]) and surface sediments from 10 regional lakes. Water chemistry, along with fork length, weight, and stable isotopes (δ 15 N, δ 13 C, δ 34 S) in fish were also measured. No lake sediments had elevated (>0.3 μg/g dw) Hg, and average Hg concentrations in fish were not sufficiently high ( 13 C), and certain lake variables (e.g., pH for YP). PS with more pelagic feeding habits had higher δ 34 S and Hg than those with more littoral feeding habits. Potential biological linkages between fish Hg and δ 34 S, a parameter that may be related to the lake sulphate-reducing bacteria activity, requires further investigation. - Fish from lakes near a localized geological Hg source do not have elevated Hg concentrations

Temperature, Frequency, and Young’s Modulus of an Aluminum Tuning Fork

Directory of Open Access Journals (Sweden)

Zachery L. Greer

2011-01-01

Full Text Available The frequency produced by a standard C (523.3 Hz aluminum alloy tuning fork when struck at temperatures ranging from 29 ̊C to 300 ̊C was studied. It was found that frequency decreased with increasing temperature with an inverse exponential relationship. The frequency was used to calculate Young’s Modulus for aluminum, with the results being in close agreement with published values.

Coupling of replisome movement with nucleosome dynamics can contribute to the parent-daughter information transfer.

Science.gov (United States)

Bameta, Tripti; Das, Dibyendu; Padinhateeri, Ranjith

2018-06-01

Positioning of nucleosomes along the genomic DNA is crucial for many cellular processes that include gene regulation and higher order packaging of chromatin. The question of how nucleosome-positioning information from a parent chromatin gets transferred to the daughter chromatin is highly intriguing. Accounting for experimentally known coupling between replisome movement and nucleosome dynamics, we propose a model that can obtain de novo nucleosome assembly similar to what is observed in recent experiments. Simulating nucleosome dynamics during replication, we argue that short pausing of the replication fork, associated with nucleosome disassembly, can be a event crucial for communicating nucleosome positioning information from parent to daughter. We show that the interplay of timescales between nucleosome disassembly (τp) at the replication fork and nucleosome sliding behind the fork (τs) can give rise to a rich 'phase diagram' having different inherited patterns of nucleosome organization. Our model predicts that only when τp ≥ τs the daughter chromatin can inherit nucleosome positioning of the parent.

Molecular electronic junction transport

DEFF Research Database (Denmark)

Solomon, Gemma C.; Herrmann, Carmen; Ratner, Mark

2012-01-01

Whenasinglemolecule,oracollectionofmolecules,isplacedbetween two electrodes and voltage is applied, one has a molecular transport junction. We discuss such junctions, their properties, their description, and some of their applications. The discussion is qualitative rather than quantitative, and f...

Gap junctions and motor behavior

DEFF Research Database (Denmark)

Kiehn, Ole; Tresch, Matthew C.

2002-01-01

The production of any motor behavior requires coordinated activity in motor neurons and premotor networks. In vertebrates, this coordination is often assumed to take place through chemical synapses. Here we review recent data suggesting that electrical gap-junction coupling plays an important role...... in coordinating and generating motor outputs in embryonic and early postnatal life. Considering the recent demonstration of a prevalent expression of gap-junction proteins and gap-junction structures in the adult mammalian spinal cord, we suggest that neuronal gap-junction coupling might also contribute...... to the production of motor behavior in adult mammals....

Ferromagnetic Josephson Junctions for Cryogenic Memory

Science.gov (United States)

Niedzielski, Bethany M.; Gingrich, Eric C.; Khasawneh, Mazin A.; Loloee, Reza; Pratt, William P., Jr.; Birge, Norman O.

2015-03-01

Josephson junctions containing ferromagnetic materials are of interest for both scientific and technological purposes. In principle, either the amplitude of the critical current or superconducting phase shift across the junction can be controlled by the relative magnetization directions of the ferromagnetic layers in the junction. Our approach concentrates on phase control utilizing two junctions in a SQUID geometry. We will report on efforts to control the phase of junctions carrying either spin-singlet or spin-triplet supercurrent for cryogenic memory applications. Supported by Northorp Grumman Corporation and by IARPA under SPAWAR Contract N66001-12-C-2017.

A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans.

Directory of Open Access Journals (Sweden)

Samantha C Lewis

2015-02-01

Full Text Available Mitochondrial DNA (mtDNA encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.

Database Replication Prototype

OpenAIRE

Vandewall, R.

2000-01-01

This report describes the design of a Replication Framework that facilitates the implementation and com-parison of database replication techniques. Furthermore, it discusses the implementation of a Database Replication Prototype and compares the performance measurements of two replication techniques based on the Atomic Broadcast communication primitive: pessimistic active replication and optimistic active replication. The main contributions of this report can be split into four parts....

Electronic noise of superconducting tunnel junction detectors

International Nuclear Information System (INIS)

Jochum, J.; Kraus, H.; Gutsche, M.; Kemmather, B.; Feilitzsch, F. v.; Moessbauer, R.L.

1994-01-01

The optimal signal to noise ratio for detectors based on superconducting tunnel junctions is calculated and compared for the cases of a detector consisting of one single tunnel junction, as well as of series and of parallel connections of such tunnel junctions. The influence of 1 / f noise and its dependence on the dynamical resistance of tunnel junctions is discussed quantitatively. A single tunnel junction yields the minimum equivalent noise charge. Such a tunnel junction exhibits the best signal to noise ratio if the signal charge is independent of detector size. In case, signal charge increases with detector size, a parallel or a series connection of tunnel junctions would provide the optimum signal to noise ratio. The equivalent noise charge and the respective signal to noise ratio are deduced as functions of tunnel junction parameters such as tunneling time, quasiparticle lifetime, etc. (orig.)

"Forked Tongue: The Politics of Bilingual Education," by Rosalie Pedalino Porter. Book Review.

Science.gov (United States)

Amabisca, Anastasia Aimee

1994-01-01

Based on personal experience rather than empirical evidence, "Forked Tongue" presents a biased view that could damage the future of bilingual education in the United States. The book considers bilingual education a threat because it prolongs the learning of English and promotes the maintenance of other "un-American" languages…

Atomic-scaled characterization of graphene PN junctions

Science.gov (United States)

Zhou, Xiaodong; Wang, Dennis; Dadgar, Ali; Agnihotri, Pratik; Lee, Ji Ung; Reuter, Mark C.; Ross, Frances M.; Pasupathy, Abhay N.

Graphene p-n junctions are essential devices for studying relativistic Klein tunneling and the Veselago lensing effect in graphene. We have successfully fabricated graphene p-n junctions using both lithographically pre-patterned substrates and the stacking of vertical heterostructures. We then use our 4-probe STM system to characterize the junctions. The ability to carry out scanning electron microscopy (SEM) in our STM instrument is essential for us to locate and measure the junction interface. We obtain both the topography and dI/dV spectra at the junction area, from which we track the shift of the graphene chemical potential with position across the junction interface. This allows us to directly measure the spatial width and roughness of the junction and its potential barrier height. We will compare the junction properties of devices fabricated by the aforementioned two methods and discuss their effects on the performance as a Veselago lens.

Chemical and biological sensing using tuning forks

Science.gov (United States)

Tao, Nongjian; Boussaad, Salah

2012-07-10

A device for sensing a chemical analyte is disclosed. The device is comprised of a vibrating structure having first and second surfaces and having an associated resonant frequency and a wire coupled between the first and second surfaces of the vibrating structure, wherein the analyte interacts with the wire and causes a change in the resonant frequency of the vibrating structure. The vibrating structure can include a tuning fork. The vibrating structure can be comprised of quartz. The wire can be comprised of polymer. A plurality of vibrating structures are arranged in an array to increase confidence by promoting a redundancy of measurement or to detect a plurality of chemical analytes. A method of making a device for sensing a chemical analyte is also disclosed.

Gaussian Random Fields Methods for Fork-Join Network with Synchronization Constraints

Science.gov (United States)

2014-12-22

substantial efforts were dedicated to the study of the max-plus recursions [21, 3, 12]. More recently, Atar et al. [2] have studied a fork-join...feedback and NES, Atar et al. [2] show that a dynamic priority discipline achieves throughput optimal- ity asymptotically in the conventional heavy...2011) Patient flow in hospitals: a data-based queueing-science perspective. Submitted to Stochastic Systems, 20. [2] R. Atar , A. Mandelbaum and A

Stability of large-area molecular junctions

NARCIS (Netherlands)

Akkerman, Hylke B.; Kronemeijer, Auke J.; Harkema, Jan; van Hal, Paul A.; Smits, Edsger C. P.; de Leeuw, Dago M.; Blom, Paul W. M.

The stability of molecular junctions is crucial for any application of molecular electronics. Degradation of molecular junctions when exposed to ambient conditions is regularly observed. In this report the stability of large-area molecular junctions under ambient conditions for more than two years

Superconducting flux qubits with π-junctions

International Nuclear Information System (INIS)

Shcherbakova, Anastasia

2014-01-01

In this thesis, we present a fabrication technology of Al/AlO x /Al Josephson junctions on Nb pads. The described technology gives the possibility of combining a variety of Nb-based superconducting circuits, like pi-junction phase-shifters with sub-micron Al/AlO x /Al junctions. Using this approach, we fabricated hybrid Nb/Al flux qubits with and without the SFS-junctions and studied dispersive magnetic field response of these qubits as well as their spectroscopy characteristics.

Resonance Transport of Graphene Nanoribbon T-Shaped Junctions

International Nuclear Information System (INIS)

Xiao-Lan, Kong; Yong-Jian, Xiong

2010-01-01

We investigate the transport properties of T-shaped junctions composed of armchair graphene nanoribbons of different widths. Three types of junction geometries are considered. The junction conductance strongly depends on the atomic features of the junction geometry. When the shoulders of the junction have zigzag type edges, sharp conductance resonances usually appear in the low energy region around the Dirac point, and a conductance gap emerges. When the shoulders of the junction have armchair type edges, the conductance resonance behavior is weakened significantly, and the metal-metal-metal junction structures show semimetallic behaviors. The contact resistance also changes notably due to the various interface geometries of the junction

The Replication Recipe: What makes for a convincing replication?

NARCIS (Netherlands)

Brandt, M.J.; IJzerman, H.; Dijksterhuis, A.J.; Farach, F.J.; Geller, J.; Giner-Sorolla, R.; Grange, J.A.; Perugini, M.; Spies, J.R.; Veer, A. van 't

2014-01-01

Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

The replication recipe : What makes for a convincing replication?

NARCIS (Netherlands)

Brandt, M.J.; IJzerman, H.; Dijksterhuis, Ap; Farach, Frank J.; Geller, Jason; Giner-Sorolla, Roger; Grange, James A.; Perugini, Marco; Spies, Jeffrey R.; van 't Veer, Anna

Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

Electrospun single crystalline fork-like K2V8O21 as high-performance cathode materials for lithium-ion batteries

Science.gov (United States)

Hao, Pengfei; Zhu, Ting; Su, Qiong; Lin, Jiande; Cui, Rong; Cao, Xinxin; Wang, Yaping; Pan, Anqiang

2018-06-01

Single crystalline fork-like potassium vanadate (K2V8O21) has been successfully prepared through electrospinning combined with a subsequent annealing process. The as-obtained K2V8O21 forks show a unique layer-by-layer stacked structure with conductive carbon. When used as cathode materials for lithium-ion batteries, the as-prepared fork-like materials exhibit high specific discharge capacity and excellent cyclic stability. High specific discharge capacity of 200.2 mA h g-1 and 131.5 mA h g-1 can be delivered at the current densities of 50 mA g-1 and 500 mA g-1, respectively. Furthermore, the K2V8O21 electrodes exhibit excellent long-term cycling stability that maintain a capacity of 108.3 mA h g-1 after 300 cycles at 500 mA g-1 with a fading rate of only 0.054% per cycle, revealing their potential applications in next generation high-performance lithium-ion batteries.

DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

Science.gov (United States)

de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

1998-08-15

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

Josephson junctions with ferromagnetic interlayer

International Nuclear Information System (INIS)

Wild, Georg Hermann

2012-01-01

We report on the fabrication of superconductor/insulator/ferromagnetic metal/superconductor (Nb/AlO x /Pd 0.82 Ni 0.18 /Nb) Josephson junctions (SIFS JJs) with high critical current densities, large normal resistance times area products, and high quality factors. For these junctions, a transition from 0- to π-coupling is observed for a thickness d F =6 nm of the ferromagnetic Pd 0.82 Ni 0.18 interlayer. The magnetic field dependence of the critical current of the junctions demonstrates good spatial homogeneity of the tunneling barrier and ferromagnetic interlayer. Magnetic characterization shows that the Pd 0.82 Ni 0.18 has an out-of-plane anisotropy and large saturation magnetization indicating negligible dead layers at the interfaces. A careful analysis of Fiske modes up to about 400 GHz provides valuable information on the junction quality factor and the relevant damping mechanisms. Whereas losses due to quasiparticle tunneling dominate at low frequencies, at high frequencies the damping is explained by the finite surface resistance of the junction electrodes. High quality factors of up to 30 around 200 GHz have been achieved. They allow to study the junction dynamics, in particular the switching probability from the zero-voltage into the voltage state with and without microwave irradiation. The experiments with microwave irradiation are well explained within semi-classical models and numerical simulations. In contrast, at mK temperature the switching dynamics without applied microwaves clearly shows secondary quantum effects. Here, we could observe for the first time macroscopic quantum tunneling in Josephson junctions with a ferromagnetic interlayer. This observation excludes fluctuations of the critical current as a consequence of an unstable magnetic domain structure of the ferromagnetic interlayer and affirms the suitability of SIFS Josephson junctions for quantum information processing.

Josephson junctions with ferromagnetic interlayer

Energy Technology Data Exchange (ETDEWEB)

Wild, Georg Hermann

2012-03-04

We report on the fabrication of superconductor/insulator/ferromagnetic metal/superconductor (Nb/AlO{sub x}/Pd{sub 0.82}Ni{sub 0.18}/Nb) Josephson junctions (SIFS JJs) with high critical current densities, large normal resistance times area products, and high quality factors. For these junctions, a transition from 0- to {pi}-coupling is observed for a thickness d{sub F}=6 nm of the ferromagnetic Pd{sub 0.82}Ni{sub 0.18} interlayer. The magnetic field dependence of the critical current of the junctions demonstrates good spatial homogeneity of the tunneling barrier and ferromagnetic interlayer. Magnetic characterization shows that the Pd{sub 0.82}Ni{sub 0.18} has an out-of-plane anisotropy and large saturation magnetization indicating negligible dead layers at the interfaces. A careful analysis of Fiske modes up to about 400 GHz provides valuable information on the junction quality factor and the relevant damping mechanisms. Whereas losses due to quasiparticle tunneling dominate at low frequencies, at high frequencies the damping is explained by the finite surface resistance of the junction electrodes. High quality factors of up to 30 around 200 GHz have been achieved. They allow to study the junction dynamics, in particular the switching probability from the zero-voltage into the voltage state with and without microwave irradiation. The experiments with microwave irradiation are well explained within semi-classical models and numerical simulations. In contrast, at mK temperature the switching dynamics without applied microwaves clearly shows secondary quantum effects. Here, we could observe for the first time macroscopic quantum tunneling in Josephson junctions with a ferromagnetic interlayer. This observation excludes fluctuations of the critical current as a consequence of an unstable magnetic domain structure of the ferromagnetic interlayer and affirms the suitability of SIFS Josephson junctions for quantum information processing.

Hydraulic, geomorphic, and trout habitat conditions of the Lake Fork of the Gunnison River in Hinsdale County, Lake City, Colorado, Water Years 2010-2011

Science.gov (United States)

Williams, Cory A.; Richards, Rodney J.; Schaffrath, Keelin R.

2015-01-01

Channel rehabilitation, or reconfiguration, to mitigate a variety of riverine problems has become a common practice in the western United States. However, additional work to monitor and assess the channel response to, and the effectiveness of, these modifications over longer periods of time (decadal or longer) is still needed. The Lake Fork of the Gunnison River has been an area of active channel modification to accommodate the needs of the Lake City community since the 1950s. The Lake Fork Valley Conservancy District began a planning process to assess restoration options for a reach of the Lake Fork in Lake City to enhance hydraulic and ecologic characteristics of the reach. Geomorphic channel form is affected by land-use changes within the basin and geologic controls within the reach. The historic channel was defined as a dynamic, braided channel with an active flood plain. This can result in a natural tendency for the channel to braid. A braided channel can affect channel stability of reconfigured reaches when a single-thread meandering channel is imposed on the stream. The U.S. Geological Survey, in cooperation with the Colorado Water Conservation Board and Colorado River Water Conservation District, began a study in 2010 to quantify existing hydraulic and habitat conditions for a reach of the Lake Fork of the Gunnison River in Lake City, Colorado. The purpose of this report is to quantify existing Lake Fork hydraulic and habitat conditions and establish a baseline against which post-reconfiguration conditions can be compared. This report (1) quantifies the existing hydraulic and geomorphic conditions in a 1.1-kilometer section of the Lake Fork at Lake City that has been proposed as a location for future channel-rehabilitation efforts, (2) characterizes the habitat suitability of the reach for two trout species based on physical conditions within the stream, and (3) characterizes the current riparian canopy density.

Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells.

Science.gov (United States)

Renaud, Emilie; Barascu, Aurelia; Rosselli, Filippo

2016-01-29

To rescue collapsed replication forks cells utilize homologous recombination (HR)-mediated mechanisms to avoid the induction of gross chromosomal abnormalities that would be generated by non-homologous end joining (NHEJ). Using DNA interstrand crosslinks as a replication barrier, we investigated how the Fanconi anemia (FA) pathway promotes HR at stalled replication forks. FA pathway inactivation results in Fanconi anemia, which is associated with a predisposition to cancer. FANCD2 monoubiquitination and assembly in subnuclear foci appear to be involved in TIP60 relocalization to the chromatin to acetylates histone H4K16 and prevents the binding of 53BP1 to its docking site, H4K20Me2. Thus, FA pathway loss-of-function results in accumulation of 53BP1, RIF1 and RAP80 at damaged chromatin, which impair DNA resection at stalled replication fork-associated DNA breaks and impede HR. Consequently, DNA repair in FA cells proceeds through the NHEJ pathway, which is likely responsible for the accumulation of chromosome abnormalities. We demonstrate that the inhibition of NHEJ or deacetylase activity rescue HR in FA cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Kinetics of [32P]orthophosphate and [3H]thymidine incorporation into newly replicating DNA in vivo

International Nuclear Information System (INIS)

Panzeter, P.; Ringer, D.

1986-01-01

Nuclear DNA can be empirically subdivided into three populations: (1) bulk DNA or low salt-soluble DNA (75%), (2) high salt-soluble DNA (23%), and (3) matrix DNA which remains tightly bound to the nuclear matrix (2%). Newly replicating DNA is associated with the nuclear matrix in regenerating rat liver. To study the incorporation of DNA precursors into replicating DNA via the salvage vs the de novo pathway, 100μCi [ 3 H]thymidine ( 3 H-Thd) and 5mCi [ 32 P]orthophosphate ( 32 P/sub i/) were injected into the hepatic portal vein of partially hepatectomized rats. Increasing time of 3 H-Thd incorporation showed the label is chased from matrix DNA to bulk DNA. After a 10 min pulse, 13% of the total specific activity is associated with bulk DNA and 57% with matrix DNA. After 30 min, 32% and 36% of the total specific activity remain associated with bulk and matrix DNA, respectively, indicating that most of the 3 H has been chased from the matrix DNA. In contrast, after injection of 32 P/sub i/, the amount of label in matrix DNA increases to a maximum at 30 min and only then begins to decrease. At 10 min the specific activity/total specific activity of bulk DNA is 7% and of matrix DNA is 66% vs 8% and 82% after 30 min. The kinetic pattern of 32 P/sub i/ incorporation differs dramatically from that of 3 H-Thd suggesting (a) the incorporation of de novo precursors lags significantly behind that of precursors entering through the salvage pathway, or (b) there may be two distinct classes of replication forks

Dynamic DNA binding, junction recognition and G4 melting activity underlie the telomeric and genome-wide roles of human CST.

Science.gov (United States)

Bhattacharjee, Anukana; Wang, Yongyao; Diao, Jiajie; Price, Carolyn M

2017-12-01

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Electron optics with ballistic graphene junctions

Science.gov (United States)

Chen, Shaowen

Electrons transmitted across a ballistic semiconductor junction undergo refraction, analogous to light rays across an optical boundary. A pn junction theoretically provides the equivalent of a negative index medium, enabling novel electron optics such as negative refraction and perfect (Veselago) lensing. In graphene, the linear dispersion and zero-gap bandstructure admit highly transparent pn junctions by simple electrostatic gating, which cannot be achieved in conventional semiconductors. Robust demonstration of these effects, however, has not been forthcoming. Here we employ transverse magnetic focusing to probe propagation across an electrostatically defined graphene junction. We find perfect agreement with the predicted Snell's law for electrons, including observation of both positive and negative refraction. Resonant transmission across the pn junction provides a direct measurement of the angle dependent transmission coefficient, and we demonstrate good agreement with theory. Comparing experimental data with simulation reveals the crucial role played by the effective junction width, providing guidance for future device design. Efforts toward sharper pn junction and possibility of zero field Veselago lensing will also be discussed. This work is supported by the Semiconductor Research Corporations NRI Center for Institute for Nanoelectronics Discovery and Exploration (INDEX).

An Inventory and Evaluation of Architectural and Engineering Resources of the Big South Fork National River and Recreation Area, Tennessee and Kentucky.

Science.gov (United States)

1982-02-25

coordinated multidisciplinary study of both the architectural and engineering resources of the National Area. Both research b1 orientation and...South Fork just north of Rugby , and traveled through the site where Jamestown, Tennessee, now stands. A third trail, the Chickamauga Path, left the...Thomas Hughes (1881), the founder of the English colony of Rugby , Tennessee, described his neighbors in the Big South Fork area as mostly poor men

Peltier cooling in molecular junctions

Science.gov (United States)

Cui, Longji; Miao, Ruijiao; Wang, Kun; Thompson, Dakotah; Zotti, Linda Angela; Cuevas, Juan Carlos; Meyhofer, Edgar; Reddy, Pramod

2018-02-01

The study of thermoelectricity in molecular junctions is of fundamental interest for the development of various technologies including cooling (refrigeration) and heat-to-electricity conversion1-4. Recent experimental progress in probing the thermopower (Seebeck effect) of molecular junctions5-9 has enabled studies of the relationship between thermoelectricity and molecular structure10,11. However, observations of Peltier cooling in molecular junctions—a critical step for establishing molecular-based refrigeration—have remained inaccessible. Here, we report direct experimental observations of Peltier cooling in molecular junctions. By integrating conducting-probe atomic force microscopy12,13 with custom-fabricated picowatt-resolution calorimetric microdevices, we created an experimental platform that enables the unified characterization of electrical, thermoelectric and energy dissipation characteristics of molecular junctions. Using this platform, we studied gold junctions with prototypical molecules (Au-biphenyl-4,4'-dithiol-Au, Au-terphenyl-4,4''-dithiol-Au and Au-4,4'-bipyridine-Au) and revealed the relationship between heating or cooling and charge transmission characteristics. Our experimental conclusions are supported by self-energy-corrected density functional theory calculations. We expect these advances to stimulate studies of both thermal and thermoelectric transport in molecular junctions where the possibility of extraordinarily efficient energy conversion has been theoretically predicted2-4,14.

Synchronous contextual irregularities affect early scene processing: replication and extension.

Science.gov (United States)

Mudrik, Liad; Shalgi, Shani; Lamy, Dominique; Deouell, Leon Y

2014-04-01

Whether contextual regularities facilitate perceptual stages of scene processing is widely debated, and empirical evidence is still inconclusive. Specifically, it was recently suggested that contextual violations affect early processing of a scene only when the incongruent object and the scene are presented a-synchronously, creating expectations. We compared event-related potentials (ERPs) evoked by scenes that depicted a person performing an action using either a congruent or an incongruent object (e.g., a man shaving with a razor or with a fork) when scene and object were presented simultaneously. We also explored the role of attention in contextual processing by using a pre-cue to direct subjects׳ attention towards or away from the congruent/incongruent object. Subjects׳ task was to determine how many hands the person in the picture used in order to perform the action. We replicated our previous findings of frontocentral negativity for incongruent scenes that started ~ 210 ms post stimulus presentation, even earlier than previously found. Surprisingly, this incongruency ERP effect was negatively correlated with the reaction times cost on incongruent scenes. The results did not allow us to draw conclusions about the role of attention in detecting the regularity, due to a weak attention manipulation. By replicating the 200-300 ms incongruity effect with a new group of subjects at even earlier latencies than previously reported, the results strengthen the evidence for contextual processing during this time window even when simultaneous presentation of the scene and object prevent the formation of prior expectations. We discuss possible methodological limitations that may account for previous failures to find this an effect, and conclude that contextual information affects object model selection processes prior to full object identification, with semantic knowledge activation stages unfolding only later on. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dynamics of pi-junction interferometer circuits

DEFF Research Database (Denmark)

Kornkev, V.K.; Mozhaev, P.B.; Borisenko, I.V.

2002-01-01

The pi-junction superconducting circuit dynamics was studied by means of numerical simulation technique. Parallel arrays consisting of Josephson junctions of both 0- and pi-type were studied as a model of high-T-c grain-boundary Josephson junction. The array dynamics and the critical current depe...

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

Science.gov (United States)

Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

2014-10-28

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

Geodynamical simulation of the RRF triple junction

Science.gov (United States)

Wang, Z.; Wei, D.; Liu, M.; Shi, Y.; Wang, S.

2017-12-01

Triple junction is the point at which three plate boundaries meet. Three plates at the triple junction form a complex geological tectonics, which is a natural laboratory to study the interactions of plates. This work studies a special triple junction, the oceanic transform fault intersects the collinear ridges with different-spreading rates, which is free of influence of ridge-transform faults and nearby hotspots. First, we build 3-D numerical model of this triple junction used to calculate the stead-state velocity and temperature fields resulting from advective and conductive heat transfer. We discuss in detail the influence of the velocity and temperature fields of the triple junction from viscosity, spreading rate of the ridge. The two sides of the oceanic transform fault are different sensitivities to the two factors. And, the influence of the velocity mainly occurs within 200km of the triple junction. Then, we modify the model by adding a ridge-transform fault to above model and directly use the velocity structure of the Macquarie triple junction. The simulation results show that the temperature at both sides of the oceanic transform fault decreases gradually from the triple junction, but the temperature difference between the two sides is a constant about 200°. And, there is little effect of upwelling velocity away from the triple junction 100km. The model results are compared with observational data. The heat flux and thermal topography along the oceanic transform fault of this model are consistent with the observed data of the Macquarie triple junction. The earthquakes are strike slip distributed along the oceanic transform fault. Their depths are also consistent with the zone of maximum shear stress. This work can help us to understand the interactions of plates of triple junctions and help us with the foundation for the future study of triple junctions.

Loss models for long Josephson junctions

DEFF Research Database (Denmark)

Olsen, O. H.; Samuelsen, Mogens Rugholm

1984-01-01

A general model for loss mechanisms in long Josephson junctions is presented. An expression for the zero-field step is found for a junction of overlap type by means of a perturbation method. Comparison between analytic solution and perturbation result shows good agreement.......A general model for loss mechanisms in long Josephson junctions is presented. An expression for the zero-field step is found for a junction of overlap type by means of a perturbation method. Comparison between analytic solution and perturbation result shows good agreement....

Dynamics of the Josephson multi-junction system with junctions characterized by non-sinusoidal current - phase relationship

International Nuclear Information System (INIS)

Abal'osheva, I.; Lewandowski, S.J.

2004-01-01

It is shown that the inclusion of junctions characterized by non-sinusoidal current - phase relationship in the systems composed of multiple Josephson junctions - results in the appearance of additional system phase states. Numerical simulations and stability considerations confirm that those phase states can be realized in practice. Moreover, spontaneous formation of the grain boundary junctions in high-T c superconductors with non-trivial current-phase relations due to the d-wave symmetry of the order parameter is probable. Switching between the phase states of multiple grain boundary junction systems can lead to additional 1/f noise in high-T c superconductors. (author)

Theoretical and experimental investigations on synchronization in many-junction arrays of HTSC Josephson junctions. Final report

International Nuclear Information System (INIS)

Seidel, P.; Heinz, E.; Pfuch, A.; Machalett, F.; Krech, W.; Basler, M.

1996-06-01

Different many-junction arrays of Josephson junctions were studied theoretically to analyse the mechanisms of synchronization, the influence of internal and external parameters and the maximal allowed spread of parameters for the single junctions. Concepts to realize arrays using standard high-T c superconductor technology were created, e.g. the new arrangement of multijunction superconducting loops (MSL). First experimental results show the relevance of this concept. Intrinsic one-dimensional arrays in thin film technology were prepared as mesas out of Bi or Tl 2212 films. to characterize HTSC Josephson junctions methods based on the analysis of microwave-induced steps were developed. (orig.) [de

Single P-N junction tandem photovoltaic device

Science.gov (United States)

Walukiewicz, Wladyslaw [Kensington, CA; Ager, III, Joel W.; Yu, Kin Man [Lafayette, CA

2011-10-18

A single P-N junction solar cell is provided having two depletion regions for charge separation while allowing the electrons and holes to recombine such that the voltages associated with both depletion regions of the solar cell will add together. The single p-n junction solar cell includes an alloy of either InGaN or InAlN formed on one side of the P-N junction with Si formed on the other side in order to produce characteristics of a two junction (2J) tandem solar cell through only a single P-N junction. A single P-N junction solar cell having tandem solar cell characteristics will achieve power conversion efficiencies exceeding 30%.

In-Field Performance Testing of the Fork Detector for Quantitative Spent Fuel Verification

International Nuclear Information System (INIS)

Gauld, Ian C.; Hu, Jianwei; De Baere, P.; Tobin, Stephen

2015-01-01

Expanding spent fuel dry storage activities worldwide are increasing demands on safeguards authorities that perform inspections. The European Atomic Energy Community (EURATOM) and the International Atomic Energy Agency (IAEA) require measurements to verify declarations when spent fuel is transferred to difficult-to-access locations, such as dry storage casks and the repositories planned in Finland and Sweden. EURATOM makes routine use of the Fork detector to obtain gross gamma and total neutron measurements during spent fuel inspections. Data analysis is performed by modules in the integrated Review and Analysis Program (iRAP) software, developed jointly by EURATOM and the IAEA. Under the framework of the US Department of Energy-EURATOM cooperation agreement, a module for automated Fork detector data analysis has been developed by Oak Ridge National Laboratory (ORNL) using the ORIGEN code from the SCALE code system and implemented in iRAP. EURATOM and ORNL recently performed measurements on 30 spent fuel assemblies at the Swedish Central Interim Storage Facility for Spent Nuclear Fuel (Clab), operated by the Swedish Nuclear Fuel and Waste Management Company (SKB). The measured assemblies represent a broad range of fuel characteristics. Neutron count rates for 15 measured pressurized water reactor assemblies are predicted with an average relative standard deviation of 4.6%, and gamma signals are predicted on average within 2.6% of the measurement. The 15 measured boiling water reactor assemblies exhibit slightly larger deviations of 5.2% for the gamma signals and 5.7% for the neutron count rates, compared to measurements. These findings suggest that with improved analysis of the measurement data, existing instruments can provide increased verification of operator declarations of the spent fuel and thereby also provide greater ability to confirm integrity of an assembly. These results support the application of the Fork detector as a fully quantitative spent fuel

In-Field Performance Testing of the Fork Detector for Quantitative Spent Fuel Verification

Energy Technology Data Exchange (ETDEWEB)

Gauld, Ian C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hu, Jianwei [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); De Baere, P. [European Commission (Luxembourg). DG Energy, Directorate Nuclear Safeguards; Vaccaro, S. [European Commission (Luxembourg). DG Energy, Directorate Nuclear Safeguards; Schwalbach, P. [European Commission (Luxembourg). DG Energy, Directorate Nuclear Safeguards; Liljenfeldt, Henrik [Swedish Nuclear Fuel and Waste Management Company (Sweden); Tobin, Stephen [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2015-01-01

Expanding spent fuel dry storage activities worldwide are increasing demands on safeguards authorities that perform inspections. The European Atomic Energy Community (EURATOM) and the International Atomic Energy Agency (IAEA) require measurements to verify declarations when spent fuel is transferred to difficult-to-access locations, such as dry storage casks and the repositories planned in Finland and Sweden. EURATOM makes routine use of the Fork detector to obtain gross gamma and total neutron measurements during spent fuel inspections. Data analysis is performed by modules in the integrated Review and Analysis Program (iRAP) software, developed jointly by EURATOM and the IAEA. Under the framework of the US Department of Energy–EURATOM cooperation agreement, a module for automated Fork detector data analysis has been developed by Oak Ridge National Laboratory (ORNL) using the ORIGEN code from the SCALE code system and implemented in iRAP. EURATOM and ORNL recently performed measurements on 30 spent fuel assemblies at the Swedish Central Interim Storage Facility for Spent Nuclear Fuel (Clab), operated by the Swedish Nuclear Fuel and Waste Management Company (SKB). The measured assemblies represent a broad range of fuel characteristics. Neutron count rates for 15 measured pressurized water reactor assemblies are predicted with an average relative standard deviation of 4.6%, and gamma signals are predicted on average within 2.6% of the measurement. The 15 measured boiling water reactor assemblies exhibit slightly larger deviations of 5.2% for the gamma signals and 5.7% for the neutron count rates, compared to measurements. These findings suggest that with improved analysis of the measurement data, existing instruments can provide increased verification of operator declarations of the spent fuel and thereby also provide greater ability to confirm integrity of an assembly. These results support the application of the Fork detector as a fully quantitative spent fuel

Lentiform fork sign: a magnetic resonance finding in a case of acute metabolic acidosis.

Science.gov (United States)

Grasso, Daniela; Borreggine, Carmela; Perfetto, Francesco; Bertozzi, Vincenzo; Trivisano, Marina; Specchio, Luigi Maria; Grilli, Gianpaolo; Macarini, Luca

2014-06-01

We report a 33 year-old woman addicted to chronic unspecified solvents abuse with stupor, respiratory disorders, tetraplegia and severe metabolic acidosis. On admission an unenhanced cranial CT scan showed symmetrical hypodensities of both lentiform nuclei. MR imaging performed 12 hours after stupor demonstrates bilateral putaminal hemorrhagic necrosis, bilateral external capsule, corona radiata and deep cerebellar hyperintensities with right cingulate cortex involvement. DWI reflected bilateral putaminal hyperintensities with restricted water diffusion as to citotoxic edema and development of vasogenic edema in the external capsule recalling a fork. On day twenty, after specific treatments MRI demonstrated a bilateral putaminal marginal enhancement. Bilateral putaminal necrosis is a characteristic but non-specific radiological finding of methanol poisoning. Lentiform Fork sign is a rare MRI finding reported in literature in 22 patients with various conditions characterized by metabolic acidosis. Vasogenic edema may be due to the differences in metabolic vulnerability between neurons and astrocytes. We postulate that metabolic acidosis could have an important role to generate this sign.

The anatomical locus of T-junction processing.

Science.gov (United States)

Schirillo, James A

2009-07-01

Inhomogeneous surrounds can produce either asymmetrical or symmetrical increment/decrement induction by orienting T-junctions to selectively group a test patch with surrounding regions [Melfi, T., & Schirillo, J. (2000). T-junctions in inhomogeneous surrounds. Vision Research, 40, 3735-3741]. The current experiments aimed to determine where T-junctions are processed by presenting each eye with a different image so that T-junctions exist only in the fused percept. Only minor differences were found between retinal and cortical versus cortical-only conditions, indicating that T-junctions are processed cortically.

Mixing in T-junctions

NARCIS (Netherlands)

Kok, Jacobus B.W.; van der Wal, S.

1996-01-01

The transport processes that are involved in the mixing of two gases in a T-junction mixer are investigated. The turbulent flow field is calculated for the T-junction with the k- turbulence model by FLOW3D. In the mathematical model the transport of species is described with a mixture fraction

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.

Science.gov (United States)

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P; Khun, Joelle; Vos, Marten H; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-05-04

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

Science.gov (United States)

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P.; Khun, Joelle; Vos, Marten H.; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-01-01

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. PMID:22431731

Flood-inundation maps for North Fork Salt Creek at Nashville, Indiana

Science.gov (United States)

Martin, Zachary W.

2017-11-13

Digital flood-inundation maps for a 3.2-mile reach of North Fork Salt Creek at Nashville, Indiana, were created by the U.S. Geological Survey (USGS) in cooperation with the Indiana Department of Transportation. The flood-inundation maps, which can be accessed through the USGS Flood Inundation Mapping Science website at http://water.usgs.gov/osw/flood_inundation/, depict estimates of the areal extent and depth of flooding that correspond to selected water levels (stages) at the North Fork Salt Creek at Nashville, Ind., streamgage (USGS station number 03371650). Real-time stages at this streamgage may be obtained from the USGS National Water Information System at http://waterdata.usgs.gov/nwis or the National Weather Service (NWS) Advanced Hydrologic Prediction Service at http:/water.weather.gov/ahps/, which also shows observed USGS stages at the same site as the USGS streamgage (NWS site NFSI3).Flood profiles were computed for the stream reach by means of a one-dimensional, step-backwater hydraulic modeling software developed by the U.S. Army Corps of Engineers. The hydraulic model was calibrated using the current (2015) stage-discharge rating at the USGS streamgage 03371650, North Fork Salt Creek at Nashville, Ind. The hydraulic model was then used to compute 12 water-surface profiles for flood stages at 1-foot (ft) intervals, except for the highest profile of 22.9 ft, referenced to the streamgage datum ranging from 12.0 ft (the NWS “action stage”) to 22.9 ft, which is the highest stage of the current (2015) USGS stage-discharge rating curve and 1.9 ft higher than the NWS “major flood stage.” The simulated water-surface profiles were then combined with a geographic information system digital elevation model (derived from light detection and ranging data having a 0.98-ft vertical accuracy and 4.9-ft horizontal resolution) to delineate the area flooded at each stage.The availability of these maps, along with information regarding current stage from the USGS

Effect of junction configurations on microdroplet formation in a T-junction microchannel

Science.gov (United States)

Lih, F. L.; Miao, J. M.

2015-03-01

This study investigates the dynamic formation process of water microdroplets in a silicon oil flow in a T-junction microchannel. Segmented water microdroplets are formed at the junction when the water flow is perpendicularly injected into the silicon oil flow in a straight rectangular microchannel. This study further presents the effects of the water flow inlet geometry on hydrodynamic characteristics of water microdroplet formation. A numerical multiphase volume of fluid (VOF) scheme is coupled to solve the unsteady three-dimensional laminar Navier-Stokes equations to depict the droplet formation phenomena at the junction. Predicted results on the length and generated frequency of the microdroplets agree well with experimental results in a T-junction microchannel with straight and flat inlets (the base model) for both fluid flows. Empirical correlations are reported between the volumetric flow ratio and the dimensionless microdroplet length or dimensionless frequency of droplet generation at a fixed capillary number of 4.7 · 10-3. The results of this study indicate a reduction in the droplet length of approximately 21% if the straight inlet for the water flow is modified to a downstream sudden contraction inlet for the water flow.

Noncontact atomic force microscopy in liquid environment with quartz tuning fork and carbon nanotube probe

DEFF Research Database (Denmark)

Kageshima, M.; Jensenius, Henriette; Dienwiebel, M.

2002-01-01

A force sensor for noncontact atomic force microscopy in liquid environment was developed by combining a multiwalled carbon nanotube (MWNT) probe with a quartz tuning fork. Solvation shells of octamethylcyclotetrasiloxane surface were detected both in the frequency shift and dissipation. Due to t...

Integrated Outcrop and Subsurface Studies of the Interwell Environment of Carbonate Reservoirs: Clear Fork (Leonaradian Age) Reservoirs, West Texas and New Mexico, Semi-Annual; SEMIANNUAL

International Nuclear Information System (INIS)

Ruppel, Stephen C.; Jennings, James W.; Laubach, Stephen E.

2001-01-01

Outcrop studies include stratigraphic and petrophysical analysis. Analysis of the detailed sequence- and cycle-scale architecture of the Clear Fork reservoir-equivalent outcrops in Apache Canyon is nearly complete. This work reveals two high-frequency transgressive-regressive sequences (HFS) in the lower Clear Fork composite depositional sequence and three HFS in the basal middle Clear Fork composite depositional sequence. A 1,800-ft transect of 1-inch-diameter samples was collected from one cycle at the Apache Canyon outcrop. The transect was sampled with 5-ft spacing, but there were some gaps due to cover and cliff, resulting in 181 samples. Permeability, porosity, and grain density were measured, and the spatial statistics are being analyzed geostatistically

Poster - Thur Eve - 57: Craniospinal irradiation with jagged-junction IMRT approach without beam edge matching for field junctions.

Science.gov (United States)

Cao, F; Ramaseshan, R; Corns, R; Harrop, S; Nuraney, N; Steiner, P; Aldridge, S; Liu, M; Carolan, H; Agranovich, A; Karva, A

2012-07-01

Craniospinal irradiation were traditionally treated the central nervous system using two or three adjacent field sets. A intensity-modulated radiotherapy (IMRT) plan (Jagged-Junction IMRT) which overcomes problems associated with field junctions and beam edge matching, improves planning and treatment setup efficiencies with homogenous target dose distribution was developed. Jagged-Junction IMRT was retrospectively planned on three patients with prescription of 36 Gy in 20 fractions and compared to conventional treatment plans. Planning target volume (PTV) included the whole brain and spinal canal to the S3 vertebral level. The plan employed three field sets, each with a unique isocentre. One field set with seven fields treated the cranium. Two field sets treated the spine, each set using three fields. Fields from adjacent sets were overlapped and the optimization process smoothly integrated the dose inside the overlapped junction. For the Jagged-Junction IMRT plans vs conventional technique, average homogeneity index equaled 0.08±0.01 vs 0.12±0.02, and conformity number equaled 0.79±0.01 vs 0.47±0.12. The 95% isodose surface covered (99.5±0.3)% of the PTV vs (98.1±2.0)%. Both Jagged-Junction IMRT plans and the conventional plans had good sparing of the organs at risk. Jagged-Junction IMRT planning provided good dose homogeneity and conformity to the target while maintaining a low dose to the organs at risk. Jagged-Junction IMRT optimization smoothly distributed dose in the junction between field sets. Since there was no beam matching, this treatment technique is less likely to produce hot or cold spots at the junction in contrast to conventional techniques. © 2012 American Association of Physicists in Medicine.

Multiplexing of adjacent vortex modes with the forked grating coupler

Science.gov (United States)

Nadovich, Christopher T.; Kosciolek, Derek J.; Crouse, David T.; Jemison, William D.

2017-08-01

For vortex fiber multiplexing to reach practical commercial viability, simple silicon photonic interfaces with vortex fiber will be required. These interfaces must support multiplexing. Toward this goal, an efficient singlefed multimode Forked Grating Coupler (FGC) for coupling two different optical vortex OAM charges to or from the TE0 and TE1 rectangular waveguide modes has been developed. A simple, apodized device implemented with e-beam lithography and a conventional dual-etch processing on SOI wafer exhibits low crosstalk and reasonable mode match. Advanced designs using this concept are expected to further improve performance.

Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.

Science.gov (United States)

Takahashi, Daisuke; Sato, Koichi; Hirayama, Emiko; Takata, Minoru; Kurumizaka, Hitoshi

2015-09-01

Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Caenorhabditis elegans HIM-18/SLX-4 interacts with SLX-1 and XPF-1 and maintains genomic integrity in the germline by processing recombination intermediates.

Science.gov (United States)

Saito, Takamune T; Youds, Jillian L; Boulton, Simon J; Colaiácovo, Monica P

2009-11-01

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR-mediated repair at stalled replication forks. A reduction in crossover recombination frequencies-accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction-support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.