Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

PubMed

Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel M A; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin A M; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-04-01

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.

PubMed

Barnes, Ryan P; Hile, Suzanne E; Lee, Marietta Y; Eckert, Kristin A

2017-09-01

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Large transcription units unify copy number variants and common fragile sites arising under replication stress.

PubMed

Wilson, Thomas E; Arlt, Martin F; Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W

2015-02-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. © 2015 Wilson et al.; Published by Cold Spring Harbor Laboratory Press.

Large transcription units unify copy number variants and common fragile sites arising under replication stress

PubMed Central

Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W.

2015-01-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. PMID:25373142

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

PubMed

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.

Experimental Replication of an Aeroengine Combustion Instability

NASA Technical Reports Server (NTRS)

Cohen, J. M.; Hibshman, J. R.; Proscia, W.; Rosfjord, T. J.; Wake, B. E.; McVey, J. B.; Lovett, J.; Ondas, M.; DeLaat, J.; Breisacher, K.

2000-01-01

Combustion instabilities in gas turbine engines are most frequently encountered during the late phases of engine development, at which point they are difficult and expensive to fix. The ability to replicate an engine-traceable combustion instability in a laboratory-scale experiment offers the opportunity to economically diagnose the problem (to determine the root cause), and to investigate solutions to the problem, such as active control. The development and validation of active combustion instability control requires that the causal dynamic processes be reproduced in experimental test facilities which can be used as a test bed for control system evaluation. This paper discusses the process through which a laboratory-scale experiment was designed to replicate an instability observed in a developmental engine. The scaling process used physically-based analyses to preserve the relevant geometric, acoustic and thermo-fluid features. The process increases the probability that results achieved in the single-nozzle experiment will be scalable to the engine.

FANCD2 limits replication stress and genome instability in cells lacking BRCA2

PubMed Central

Buffa, Francesca M.; McDermott, Ultan; Tarsounas, Madalena

2016-01-01

The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication fork progression and genomic instability. Our results identify a novel role for FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, which impacts on cell survival and treatment responses. PMID:27322732

Genomic Instability in Human Pluripotent Stem Cells Arises from Replicative Stress and Chromosome Condensation Defects.

PubMed

Lamm, Noa; Ben-David, Uri; Golan-Lev, Tamar; Storchová, Zuzana; Benvenisty, Nissim; Kerem, Batsheva

2016-02-04

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations such as aneuploidy in culture. These aberrations progressively increase over time and may compromise the properties and clinical utility of the cells. The underlying mechanisms that drive initial genomic instability and its continued progression are largely unknown. Here, we show that aneuploid hPSCs undergo DNA replication stress, resulting in defective chromosome condensation and segregation. Aneuploid hPSCs show altered levels of actin cytoskeletal genes controlled by the transcription factor SRF, and overexpression of SRF rescues impaired chromosome condensation and segregation defects in aneuploid hPSCs. Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate during initiation of instability in diploid cells. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis

PubMed Central

Bergoglio, Valérie; Boyer, Anne-Sophie; Walsh, Erin; Naim, Valeria; Legube, Gaëlle; Lee, Marietta Y.W.T.; Rey, Laurie; Rosselli, Filippo; Cazaux, Christophe; Eckert, Kristin A.

2013-01-01

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation–induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role. We demonstrated that Pol η accumulated at CFSs upon partial replication stress and could efficiently replicate non-B DNA sequences within CFSs. Pol η deficiency led to persistence of checkpoint-blind under-replicated CFS regions in mitosis, detectable as FANCD2-associated chromosomal sites that were transmitted to daughter cells in 53BP1-shielded nuclear bodies. Expression of a catalytically inactive mutant of Pol η increased replication fork stalling and activated the replication checkpoint. These data are consistent with the requirement of Pol η–dependent DNA synthesis during S phase at replication forks stalled in CFS regions to suppress CFS instability by preventing checkpoint-blind under-replicated DNA in mitosis. PMID:23609533

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

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

Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

DOE PAGES

Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; ...

2015-09-08

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

Mouse embryonic stem cells have increased capacity for replication fork restart driven by the specific Filia-Floped protein complex.

PubMed

Zhao, Bo; Zhang, Weidao; Cun, Yixian; Li, Jingzheng; Liu, Yan; Gao, Jing; Zhu, Hongwen; Zhou, Hu; Zhang, Rugang; Zheng, Ping

2018-01-01

Pluripotent stem cells (PSCs) harbor constitutive DNA replication stress during their rapid proliferation and the consequent genome instability hampers their applications in regenerative medicine. It is therefore important to understand the regulatory mechanisms of replication stress response in PSCs. Here, we report that mouse embryonic stem cells (ESCs) are superior to differentiated cells in resolving replication stress. Specifically, ESCs utilize a unique Filia-Floped protein complex-dependent mechanism to efficiently promote the restart of stalled replication forks, therefore maintaining genomic stability. The ESC-specific Filia-Floped complex resides on replication forks under normal conditions. Replication stress stimulates their recruitment to stalling forks and the serine 151 residue of Filia is phosphorylated in an ATR-dependent manner. This modification enables the Filia-Floped complex to act as a functional scaffold, which then promotes the stalling fork restart through a dual mechanism: both enhancing recruitment of the replication fork restart protein, Blm, and stimulating ATR kinase activation. In the Blm pathway, the scaffolds recruit the E3 ubiquitin ligase, Trim25, to the stalled replication forks, and in turn Trim25 tethers and concentrates Blm at stalled replication forks through ubiquitination. In differentiated cells, the recruitment of the Trim25-Blm complex to replication forks and the activation of ATR signaling are much less robust due to lack of the ESC-specific Filia-Floped scaffold. Thus, our study reveals that ESCs utilize an additional and unique regulatory layer to efficiently promote the stalled fork restart and maintain genomic stability.

Replication Stress and Mitotic Dysfunction in Cells Expressing Simian Virus 40 Large T Antigen

PubMed Central

Hu, Liang; Filippakis, Harilaos; Huang, Haomin; Yen, Timothy J.

2013-01-01

We previously demonstrated that simian virus 40 (SV40) large T antigen (LT) binds to the Bub1 kinase, a key regulator of the spindle checkpoint and chromosome segregation. Bub1 mutations or altered expression patterns are linked to chromosome missegregation and are considered to be a driving force in some human cancers. Here we report that LT, dependent on Bub1 binding, causes micronuclei, lagging chromatin, and anaphase bridges, which are hallmarks of chromosomal instability (CIN) and Bub1 insufficiency. Using time-lapse microscopy, we demonstrate that LT imposes a Bub1 binding-dependent delay in the metaphase-to-anaphase transition. Kinetochore fibers reveal that LT, via Bub1 binding, causes aberrant kinetochore (KT)-microtubule (MT) attachments and a shortened interkinetochore distance, consistent with a lack of tension. Previously, we showed that LT also induces the DNA damage response (DDR) via Bub1 binding. Using inducible LT cell lines, we show that an activated DDR was observed before the appearance of anaphase bridges and micronuclei. Furthermore, LT induction in serum-starved cells demonstrated γ-H2AX accumulation in cells that had not yet entered mitosis. Thus, DDR activation can occur independently of chromosome segregation defects. Replication stress pathways may be responsible, because signatures of replication stress were observed, which were attenuated by exogenous supplementation with nucleosides. Our observations allow us to propose a model that explains and integrates the diverse manifestations of genomic instability induced by LT. PMID:24067972

DNA Replication Origins and Fork Progression at Mammalian Telomeres

PubMed Central

Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

2017-01-01

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

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

PubMed

Lindstrom, Derek L; Leverich, Christina K; Henderson, Kiersten A; Gottschling, Daniel E

2011-03-01

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.

XRN2 Links Transcription Termination to DNA Damage and Replication Stress

PubMed Central

Patidar, Praveen L.; Motea, Edward A.; Dang, Tuyen T.; Manley, James L.

2016-01-01

XRN2 is a 5’-3’ exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability. PMID:27437695

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

PubMed

Morales, Julio C; Richard, Patricia; Patidar, Praveen L; Motea, Edward A; Dang, Tuyen T; Manley, James L; Boothman, David A

2016-07-01

XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

DNA replication stress as a hallmark of cancer.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2015-01-01

Human cancers share properties referred to as hallmarks, among which sustained proliferation, escape from apoptosis, and genomic instability are the most pervasive. The sustained proliferation hallmark can be explained by mutations in oncogenes and tumor suppressors that regulate cell growth, whereas the escape from apoptosis hallmark can be explained by mutations in the TP53, ATM, or MDM2 genes. A model to explain the presence of the three hallmarks listed above, as well as the patterns of genomic instability observed in human cancers, proposes that the genes driving cell proliferation induce DNA replication stress, which, in turn, generates genomic instability and selects for escape from apoptosis. Here, we review the data that support this model, as well as the mechanisms by which oncogenes induce replication stress. Further, we argue that DNA replication stress should be considered as a hallmark of cancer because it likely drives cancer development and is very prevalent.

Transcription and replication: breaking the rules of the road causes genomic instability.

PubMed

Poveda, Ana Maria; Le Clech, Mikael; Pasero, Philippe

2010-01-01

Replication and transcription machineries progress at high speed on the same DNA template, which inevitably causes traffic accidents. Problems are not only caused by frontal collisions between polymerases, but also by cotranscriptional R-loops. These RNA-DNA hybrids induce genomic instability by blocking fork progression and could be implicated in the development of cancer.

Links between DNA Replication, Stem Cells and Cancer

PubMed Central

Vassilev, Alex; DePamphilis, Melvin L.

2017-01-01

Cancers can be categorized into two groups: those whose frequency increases with age, and those resulting from errors during mammalian development. The first group is linked to DNA replication through the accumulation of genetic mutations that occur during proliferation of developmentally acquired stem cells that give rise to and maintain tissues and organs. These mutations, which result from DNA replication errors as well as environmental insults, fall into two categories; cancer driver mutations that initiate carcinogenesis and genome destabilizing mutations that promote aneuploidy through excess genome duplication and chromatid missegregation. Increased genome instability results in accelerated clonal evolution leading to the appearance of more aggressive clones with increased drug resistance. The second group of cancers, termed germ cell neoplasia, results from the mislocation of pluripotent stem cells during early development. During normal development, pluripotent stem cells that originate in early embryos give rise to all of the cell lineages in the embryo and adult, but when they mislocate to ectopic sites, they produce tumors. Remarkably, pluripotent stem cells, like many cancer cells, depend on the Geminin protein to prevent excess DNA replication from triggering DNA damage-dependent apoptosis. This link between the control of DNA replication during early development and germ cell neoplasia reveals Geminin as a potential chemotherapeutic target in the eradication of cancer progenitor cells. PMID:28125050

The Fanconi anemia pathway promotes replication-dependent DNA interstrand crosslink repair

PubMed Central

Knipscheer, Puck; Räschle, Markus; Smogorzewska, Agata; Enoiu, Milica; Ho, The Vinh; Schärer, Orlando D.; Elledge, Stephen J.; Walter, Johannes C.

2010-01-01

Fanconi anemia is a human cancer predisposition syndrome caused by mutations in thirteen Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand crosslinks (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. We make use of a cell-free system to show that the FANCI-FANCD2 complex is required for replication-dependent ICL repair. Removal of FANCD2 from extracts inhibits nucleolytic incisions near the ICL as well as translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised. PMID:19965384

SnapShot: Fanconi anemia and associated proteins.

PubMed

Wang, Anderson T; Smogorzewska, Agata

2015-01-15

Fanconi anemia is a genetic disorder resulting from biallelic mutations in one of the 17 FANC genes. It is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. The underlying cause is genomic instability resulting from the deficiency in replication-dependent DNA interstrand crosslink repair pathway commonly referred to as the Fanconi anemia-BRCA pathway. This SnapShot presents the key factors involved. Copyright © 2015 Elsevier Inc. All rights reserved.

The Zombie Instability: Using Numerical Simulation to Design a Laboratory Experiment

NASA Astrophysics Data System (ADS)

Wang, Meng; Pei, Suyang; Jiang, Chung-Hsiang; Hassanzadeh, Pedram; Marcus, Philip

2014-11-01

A new type of finite amplitude-instability has been found in numerical simulations of stratified, rotating, shear flows. The instability occurs via baroclinic critical layers that create linearly unstable vortex layers, which roll-up into vortices. Under the right conditions, those vortices can form a new generation of vortices, resulting in ``vortex self-replication'' that fills the fluid with vortices. Creating this instability in a laboratory would provide further evidence for the existence of the instability, which we first found in numerical simulations of protoplanetary disks. To design a laboratory experiment we need to know how the flow parameters-- shear, rotation and stratification, etc. affect the instability. To build an experiment economically, we also need to know how the finite-amplitude trigger of the instability scales with viscosity and the size of the domain. In this talk, we summarize our findings. We present a map, in terms of the experimentally controllable parameters, that shows where the instability occurs and whether the instability creates a few isolated transient vortices, a few long-lived vortices, or long-lived, self-replicating vortices that fill the entire flow.

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

PubMed

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.

An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism.

PubMed

Hodroj, Dana; Recolin, Bénédicte; Serhal, Kamar; Martinez, Susan; Tsanov, Nikolay; Abou Merhi, Raghida; Maiorano, Domenico

2017-05-02

Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops in vitro via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19 operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells. © 2017 The Authors.

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae.

PubMed

Kaniak-Golik, Aneta; Kuberska, Renata; Dzierzbicki, Piotr; Sledziewska-Gojska, Ewa

2017-01-01

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae

PubMed Central

Dzierzbicki, Piotr

2017-01-01

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria. PMID:28678842

Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells

PubMed Central

Yoneshima, Yasuto; Abolhassani, Nona; Iyama, Teruaki; Sakumi, Kunihiko; Shiomi, Naoko; Mori, Masahiko; Shiomi, Tadahiro; Noda, Tetsuo; Tsuchimoto, Daisuke; Nakabeppu, Yusaku

2016-01-01

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. PMID:27618981

Rescue from replication stress during mitosis.

PubMed

Fragkos, Michalis; Naim, Valeria

2017-04-03

Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease.

Rescue from replication stress during mitosis

PubMed Central

Naim, Valeria

2017-01-01

ABSTRACT Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease. PMID:28166452

Faithful replication of grating patterns in polymer through electrohydrodynamic instabilities

NASA Astrophysics Data System (ADS)

Li, H.; Yu, W.; Wang, T.; Zhang, H.; Cao, Y.; Abraham, E.; Desmulliez, M. P. Y.

2014-07-01

Electrohydrodynamic instability patterning (EHDIP) as an alternative patterning method has attracted a great deal of attention over the past decade. This article demonstrates the faithful transfer of patterns with a high aspect ratio onto a polymer film via electrohydrodynamic instabilities for a given patterned grating mask. We perform a simple mathematical analysis to determine the influence of process parameters on the pressure difference ▵P. Through numerical simulation, it is demonstrated that thick films subject to large electric fields are essential to realize this faithful replication. In particular, the influence of the material properties of the polymer on pattern replication is discussed in detail. It is found that, to achieve the smaller periodic patterns with a higher resolution, film with a larger value of the dielectric constant and smaller value of the surface tension should be chosen. In addition, an ideal replication of the mask pattern with a short evolution time is possible by reducing the viscosity of the polymer liquid. Finally, the experiments of the pattern replication with and without defects are demonstrated to compare with the numerical simulation results. It is found that experiments are in good agreement with the simulation results and prove that the numerical simulation method provides an effective way to predict faithful replication.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

PubMed

Boege, Yannick; Malehmir, Mohsen; Healy, Marc E; Bettermann, Kira; Lorentzen, Anna; Vucur, Mihael; Ahuja, Akshay K; Böhm, Friederike; Mertens, Joachim C; Shimizu, Yutaka; Frick, Lukas; Remouchamps, Caroline; Mutreja, Karun; Kähne, Thilo; Sundaravinayagam, Devakumar; Wolf, Monika J; Rehrauer, Hubert; Koppe, Christiane; Speicher, Tobias; Padrissa-Altés, Susagna; Maire, Renaud; Schattenberg, Jörn M; Jeong, Ju-Seong; Liu, Lei; Zwirner, Stefan; Boger, Regina; Hüser, Norbert; Davis, Roger J; Müllhaupt, Beat; Moch, Holger; Schulze-Bergkamen, Henning; Clavien, Pierre-Alain; Werner, Sabine; Borsig, Lubor; Luther, Sanjiv A; Jost, Philipp J; Weinlich, Ricardo; Unger, Kristian; Behrens, Axel; Hillert, Laura; Dillon, Christopher; Di Virgilio, Michela; Wallach, David; Dejardin, Emmanuel; Zender, Lars; Naumann, Michael; Walczak, Henning; Green, Douglas R; Lopes, Massimo; Lavrik, Inna; Luedde, Tom; Heikenwalder, Mathias; Weber, Achim

2017-09-11

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

PubMed Central

Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

2013-01-01

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

Increased global transcription activity as a mechanism of replication stress in cancer

PubMed Central

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M.; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-01-01

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer. PMID:27725641

Increased global transcription activity as a mechanism of replication stress in cancer.

PubMed

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-10-11

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRAS V12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRAS V12 , elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

Effects of Replication and Transcription on DNA Structure-Related Genetic Instability.

PubMed

Wang, Guliang; Vasquez, Karen M

2017-01-05

Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease.

Effects of Replication and Transcription on DNA Structure-Related Genetic Instability

PubMed Central

Wang, Guliang; Vasquez, Karen M.

2017-01-01

Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease. PMID:28067787

DNA replication stress and cancer chemotherapy.

PubMed

Kitao, Hiroyuki; Iimori, Makoto; Kataoka, Yuki; Wakasa, Takeshi; Tokunaga, Eriko; Saeki, Hiroshi; Oki, Eiji; Maehara, Yoshihiko

2018-02-01

DNA replication is one of the fundamental biological processes in which dysregulation can cause genome instability. This instability is one of the hallmarks of cancer and confers genetic diversity during tumorigenesis. Numerous experimental and clinical studies have indicated that most tumors have experienced and overcome the stresses caused by the perturbation of DNA replication, which is also referred to as DNA replication stress (DRS). When we consider therapeutic approaches for tumors, it is important to exploit the differences in DRS between tumor and normal cells. In this review, we introduce the current understanding of DRS in tumors and discuss the underlying mechanism of cancer therapy from the aspect of DRS. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

APC/C--the master controller of origin licensing?

PubMed

Sivaprasad, Umasundari; Machida, Yuichi J; Dutta, Anindya

2007-02-23

DNA replication must be tightly controlled to prevent initiation of a second round of replication until mitosis is complete. So far, components of the pre-replicative complex (Cdt1, Cdc6 and geminin) were considered key players in this regulation. In a new study, Machida and Dutta have shown that depletion of Emi1 caused cells to replicate their DNA more than once per cell cycle 1. This effect was dependent on the ability of Emi1 to inhibit the APC/C. In addition to its role in regulating entry into mitosis, oscillation of APC/C activity regulates pre-RC formation: high APC/C activity in late M/G1 allows pre-RC formation and low APC/C activity in S/G2 prevents pre-RC formation for a second time thereby preventing rereplication. Each redundant pathway to prevent rereplication is dependent on regulating one of the pre-RC components, and all of the pathways are co-regulated by Emi1 through the APC/C. In this commentary we discuss how this new role of Emi1 adds to our understanding of the regulation of replication initiation. We also review the literature to analyze whether APC/C has a role in regulating endoreduplication (a normal state of polyploidy in some differentiated cells). Similarly a role of premature APC/C activation in genomic instability of tumors is discussed.

Multiple functions of the S-phase checkpoint mediator.

PubMed

Tanaka, Katsunori

2010-01-01

There is mounting evidence that replication defects are the major source of spontaneous genomic instability in cells, and that S-phase checkpoints are the principal defense against such instability. The S-phase checkpoint mediator protein Mrc1/Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of effector kinase by a sensor kinase. In this review, the multiple functions and the regulation of the S-phase checkpoint mediator are discussed.

RECQ-like helicases Sgs1 and BLM regulate R-loop–associated genome instability

PubMed Central

Chang, Emily Yun-Chia; Novoa, Carolina A.; Aristizabal, Maria J.; Coulombe, Yan; Segovia, Romulo; Shen, Yaoqing; Keong, Christelle; Tam, Annie S.; Jones, Steven J.M.; Masson, Jean-Yves; Kobor, Michael S.

2017-01-01

Sgs1, the orthologue of human Bloom’s syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription–replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1Δ reveals copy number changes flanked by repetitive regions with high R-loop–forming potential. Analysis of BLM in Bloom’s syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop–associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop–mediated genome instability. PMID:29042409

RECQ-like helicases Sgs1 and BLM regulate R-loop-associated genome instability.

PubMed

Chang, Emily Yun-Chia; Novoa, Carolina A; Aristizabal, Maria J; Coulombe, Yan; Segovia, Romulo; Chaturvedi, Richa; Shen, Yaoqing; Keong, Christelle; Tam, Annie S; Jones, Steven J M; Masson, Jean-Yves; Kobor, Michael S; Stirling, Peter C

2017-12-04

Sgs1, the orthologue of human Bloom's syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription-replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1 Δ reveals copy number changes flanked by repetitive regions with high R-loop-forming potential. Analysis of BLM in Bloom's syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop-associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop-mediated genome instability. © 2017 Chang et al.

Role of DNA Replication Defects in Breast Cancer

DTIC Science & Technology

2009-10-01

Several recent studies have indicated that decreased levels of the MCM2-7 DNA replication proteins can lead to genomic instability (GIN) and cancer...exceeding that required for DNA replication under normal circumstances, we found that heterozygosity for 2 or more different MCMs caused genomic

The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation

PubMed Central

Galick, Heather A.; Marsden, Carolyn G.; Kathe, Scott; Dragon, Julie A.; Volk, Lindsay; Nemec, Antonia A.; Wallace, Susan S.; Prakash, Aishwarya; Doublié, Sylvie; Sweasy, Joann B.

2017-01-01

Base excision repair (BER) is a key genome maintenance pathway. The NEIL1 DNA glycosylase recognizes oxidized bases, and likely removes damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene is a rare human germline variant that encodes the NEIL1 G83D protein, which is devoid of DNA glycosylase activity. Here we show that expression of G83D NEIL1 in MCF10A immortalized but non-transformed mammary epithelial cells leads to replication fork stress. Upon treatment with hydrogen peroxide, we observe increased levels of stalled replication forks in cells expressing G83D NEIL1 versus cells expressing the wild-type (WT) protein. Double-strand breaks (DSBs) arise in G83D-expressing cells during the S and G2/M phases of the cell cycle. Interestingly, these breaks result in genomic instability in the form of high levels of chromosomal aberrations and micronuclei. Cells expressing G83D also grow in an anchorage independent manner, suggesting that the genomic instability results in a carcinogenic phenotype. Our results are consistent with the idea that an inability to remove oxidative damage in an efficient manner at the replication fork leads to genomic instability and mutagenesis. We suggest that individuals who harbor the G83D NEIL1 variant face an increased risk for human cancer. PMID:29156764

The Human RNA Polymerase I Transcription Terminator Complex Acts as a Replication Fork Barrier That Coordinates the Progress of Replication with rRNA Transcription Activity.

PubMed

Akamatsu, Yufuko; Kobayashi, Takehiko

2015-05-01

In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

PubMed

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.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2006-04-01

replication in yeast cells. In the prior reporting period we demonstrated that re-replication induces a rapid and significant decrease in cell viability...repair, DNA replication, checkpoint, cell cycle, yeast , RAD9 16. SECURITY CLASSIFICATION OF: 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON...initiation, our laboratory has been able to conditionally induce varying amounts of re- replication in yeast cells. Effectively, cells enter, but do not

Causes and Consequences of Replication Stress

PubMed Central

Zeman, Michelle K.; Cimprich, Karlene A.

2015-01-01

Replication stress is a complex phenomenon which has serious implications for genome stability, cell survival, and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATM- and Rad3-related (ATR). ATR and its downstream effectors stabilize and help to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding these pathways may be key to diagnosis and treatment of human diseases caused by defective responses to replication stress. PMID:24366029

Knockdown of RMI1 impairs DNA repair under DNA replication stress.

PubMed

Xu, Chang; Fang, Lianying; Kong, Yangyang; Xiao, Changyan; Yang, Mengmeng; Du, Li-Qing; Liu, Qiang

2017-12-09

RMI1 (RecQ-mediated genome instability protein 1) forms a conserved BTR complex with BLM, Topo IIIα, and RMI2, and its absence causes genome instability. It has been revealed that RMI1 localizes to nuclear foci with BLM and Topo IIIα in response to replication stress, and that RMI1 functions downstream of BLM in promoting replication elongation. However, the precise functions of RMI1 during replication stress are not completely understood. Here we report that RMI1 knockdown cells are hypersensitive to hydroxyurea (HU). Using comet assay, we show that RMI1 knockdown cells exhibit accumulation of broken DNAs after being released from HU treatment. Moreover, we demonstrate that RMI1 facilitates the recovery from activated checkpoint and resuming the cell cycle after replicative stress. Surprisingly, loss of RMI1 results in a failure of RAD51 loading onto DNA damage sites. These findings reveal the importance of RMI1 in response to replication stress, which could explain the molecular basis for its function in maintaining genome integrity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Temperature-dependent instability of the cTnI subunit in NIST SRM2921 characterized by tryptic peptide mapping.

PubMed

van der Burgt, Yuri E M; Cobbaert, Christa M; Dalebout, Hans; Smit, Nico; Deelder, André M

2012-08-01

In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC-MS/MS. From a Mascot database search in combination with "semiTrypsin" tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37°C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of "unexpected" peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a "single-step" MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed. Copyright © 2012 Elsevier B.V. All rights reserved.

Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site.

PubMed

Letessier, Anne; Millot, Gaël A; Koundrioukoff, Stéphane; Lachagès, Anne-Marie; Vogt, Nicolas; Hansen, R Scott; Malfoy, Bernard; Brison, Olivier; Debatisse, Michelle

2011-02-03

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.

Single-stranded DNA-binding Protein in Vitro Eliminates the Orientation-dependent Impediment to Polymerase Passage on CAG/CTG Repeats*

PubMed Central

Delagoutte, Emmanuelle; Goellner, Geoffrey M.; Guo, Jie; Baldacci, Giuseppe; McMurray, Cynthia T.

2008-01-01

Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5′-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5′-CAG and 5′-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5′-CAG is the template. We propose a “template-push” model in which the slow speed of DNA polymerase across the 5′-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over. PMID:18263578

The Role of the Transcriptional Response to DNA Replication Stress

PubMed Central

Herlihy, Anna E.; de Bruin, Robertus A.M.

2017-01-01

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage. PMID:28257104

The Role of the Transcriptional Response to DNA Replication Stress.

PubMed

Herlihy, Anna E; de Bruin, Robertus A M

2017-03-02

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage.

Functions of Replication Protein A as a Sensor of R Loops and a Regulator of RNaseH1

PubMed Central

Nguyen, Hai Dang; Yadav, Tribhuwan; Giri, Sumanprava; Saez, Borja; Graubert, Timothy A.; Zou, Lee

2017-01-01

R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here, we show that replication protein A (RPA), a ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability. PMID:28257700

The pathobiology and mechanisms of infection of HPV.

PubMed

Wood, N H; Khammissa, R A G; Chikte, U M E; Meyerov, R; Lemmer, J; Feller, L

2010-04-01

There are more than 120 types of low-risk and high-risk human papillomaviruses, all of which are epitheliotropic. HPV infection may be latent, or active in a subclinical form or a symptomatic form, the latter manifesting as benign or malignant neoplasms. In basal cells with non-productive HPV infection some early HPV proteins are expressed independently of cell maturation: the productive cycle of HPV replication depends upon specific cellular factors of the maturation of the infected keratinocytes. In HPV-mediated oncogenesis, the combined pathobiological effects of E6 and E7 oncoproteins of high-risk HPV culminate in cellular genomic instability and transformation of persistently infected cells, that progress to the development of a malignant phenotype. In this article we provide insights into the stages of HPV infection, and into the viral genomic organization and replicative cycle.

The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair.

PubMed

Knipscheer, Puck; Räschle, Markus; Smogorzewska, Agata; Enoiu, Milica; Ho, The Vinh; Schärer, Orlando D; Elledge, Stephen J; Walter, Johannes C

2009-12-18

Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.

Both High-Fidelity Replicative and Low-Fidelity Y-Family Polymerases Are Involved in DNA Rereplication

PubMed Central

Sekimoto, Takayuki; Oda, Tsukasa; Kurashima, Kiminori; Hanaoka, Fumio

2014-01-01

DNA rereplication is a major form of aberrant replication that causes genomic instabilities, such as gene amplification. However, little is known about which DNA polymerases are involved in the process. Here, we report that low-fidelity Y-family polymerases (Y-Pols), Pol η, Pol ι, Pol κ, and REV1, significantly contribute to DNA synthesis during rereplication, while the replicative polymerases, Pol δ and Pol ε, play an important role in rereplication, as expected. When rereplication was induced by depletion of geminin, these polymerases were recruited to rereplication sites in human cell lines. This finding was supported by RNA interference (RNAi)-mediated knockdown of the polymerases, which suppressed rereplication induced by geminin depletion. Interestingly, epistatic analysis indicated that Y-Pols collaborate in a common pathway, independently of replicative polymerases. We also provide evidence for a catalytic role for Pol η and the involvement of Pol η and Pol κ in cyclin E-induced rereplication. Collectively, our findings indicate that, unlike normal S-phase replication, rereplication induced by geminin depletion and oncogene activation requires significant contributions of both Y-Pols and replicative polymerases. These findings offer important mechanistic insights into cancer genomic instability. PMID:25487575

Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

PubMed

Teng, Fang-Yuan; Hou, Xi-Miao; Fan, San-Hong; Rety, Stephane; Dou, Shuo-Xing; Xi, Xu-Guang

2017-12-01

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli. © 2017 Federation of European Biochemical Societies.

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

PubMed

Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

2017-01-01

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016 Elsevier B.V. All rights reserved.

TopoIIα prevents telomere fragility and formation of ultra thin DNA bridges during mitosis through TRF1-dependent binding to telomeres.

PubMed

d'Alcontres, Martina Stagno; Palacios, Jose Alejandro; Mejias, Diego; Blasco, Maria A

2014-01-01

Telomeres are repetitive nucleoprotein structures at the ends of chromosomes. Like most genomic regions consisting of repetitive DNA, telomeres are fragile sites prone to replication fork stalling and generation of chromosomal instability. In particular, abrogation of the TRF1 telomere binding protein leads to stalled replication forks and aberrant telomere structures known as "multitelomeric signals". Here, we report that TRF1 deficiency also leads to the formation of "ultra-fine bridges" (UFB) during mitosis, and to an increased time to complete mitosis mediated by the spindle assembly checkpoint proteins (SAC). We find that topoisomerase IIα (TopoIIα), an enzyme essential for resolution of DNA replication intermediates, binds telomeres in a TRF1-mediated manner. Indeed, similar to TRF1 abrogation, TopoIIα downregulation leads to telomere fragility and UFB, suggesting that these phenotypes are due to decreased TopoIIα at telomeres. We find that SAC proteins bind telomeres in vivo, and that this is disrupted upon TRF1 deletion. These findings suggest that TRF1 links TopoIIα and SAC proteins in a pathway that ensures correct telomere replication and mitotic segregation, unveiling how TRF1 protects from telomere fragility and mitotic defects.

Absence of MutSbeta leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

PubMed Central

Slean, Meghan M.; Panigrahi, Gagan B.; Castel, Arturo López; Pearson, August B.; Tomkinson, Alan E.; Pearson, Christopher E.

2016-01-01

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats. PMID:27155933

Mars’ Growth Stunted by an Early Giant Planet Instability

NASA Astrophysics Data System (ADS)

Clement, Matthew; Kaib, Nathan A.; Raymond, Sean N.; Walsh, Kevin J.

2017-10-01

Many dynamical aspects of the solar system can be explained by the outer planets experiencing a period of orbital instability. Though often correlated with a perceived delayed spike in the lunar cratering record known as the Late Heavy Bombardment (LHB), recent work suggests that this event may have occurred during the epoch of terrestrial planet formation. Though current simulations of terrestrial accretion can reproduce many observed qualities of the solar system, replicating the small mass of Mars requires modification to standard planet formation models. Here we use direct numerical simulations to show that an early instability in the outer solar system regularly yields properly sized Mars analogues. In 80% of simulations, we produce a Mars of the appropriate mass. Our most successful outcomes occur when the terrestrial planets evolve 10 million years (Myr), and accrete several Mars sized embryos in the Mars forming region before the instability takes place. Mars is left behind as a stranded embryo, while the remainder of these bodies are either ejected from the system or scattered towards the inner solar system where they deliver water to Earth. An early giant planet instability can thus replicate both the inner and outer solar system in a single model.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2007-04-01

Conway, A., Lockhart, D. J., Davis, R. W., Brewer , B. J., and Fangman, W. L. (2001). Replication dynamics of the yeast genome. Science 294, 115–121... Brewer , B. J. (2001). An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Mol. Cell 7, 705–713. Vas, A., Mok, W., and...replication in yeast cells. We have demonstrated that re-replication induces a rapid and significant decrease in cell viability and a cellular DNA damage

5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

PubMed Central

Delagoutte, Emmanuelle; Baldacci, Giuseppe

2011-01-01

Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs. PMID:22096622

Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome

PubMed Central

Suhasini, Avvaru N; Rawtani, Nina A; Wu, Yuliang; Sommers, Joshua A; Sharma, Sudha; Mosedale, Georgina; North, Phillip S; Cantor, Sharon B; Hickson, Ian D; Brosh, Robert M

2011-01-01

Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ–BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an ‘undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress. PMID:21240188

DUB3 and USP7 de-ubiquitinating enzymes control replication inhibitor Geminin: molecular characterization and associations with breast cancer.

PubMed

Hernández-Pérez, S; Cabrera, E; Salido, E; Lim, M; Reid, L; Lakhani, S R; Khanna, K K; Saunus, J M; Freire, R

2017-08-17

Correct control of DNA replication is crucial to maintain genomic stability in dividing cells. Inappropriate re-licensing of replicated origins is associated with chromosomal instability (CIN), a hallmark of cancer progression that at the same time provides potential opportunities for therapeutic intervention. Geminin is a critical inhibitor of the DNA replication licensing factor Cdt1. To properly achieve its functions, Geminin levels are tightly regulated through the cell cycle by ubiquitin-dependent proteasomal degradation, but the de-ubiquitinating enzymes (DUBs) involved had not been identified. Here we report that DUB3 and USP7 control human Geminin. Overexpression of either DUB3 or USP7 increases Geminin levels through reduced ubiquitination. Conversely, depletion of DUB3 or USP7 reduces Geminin levels, and DUB3 knockdown increases re-replication events, analogous to the effect of Geminin depletion. In exploring potential clinical implications, we found that USP7 and Geminin are strongly correlated in a cohort of invasive breast cancers (P<1.01E-08). As expected, Geminin expression is highly prognostic. Interestingly, we found a non-monotonic relationship between USP7 and breast cancer-specific survival, with both very low or high levels of USP7 associated with poor outcome, independent of estrogen receptor status. Altogether, our data identify DUB3 and USP7 as factors that regulate DNA replication by controlling Geminin protein stability, and suggest that USP7 may be involved in Geminin dysregulation during breast cancer progression.

PTEN in the maintenance of genome integrity: From DNA replication to chromosome segregation.

PubMed

Hou, Sheng-Qi; Ouyang, Meng; Brandmaier, Andrew; Hao, Hongbo; Shen, Wen H

2017-10-01

Faithful DNA replication and accurate chromosome segregation are the key machineries of genetic transmission. Disruption of these processes represents a hallmark of cancer and often results from loss of tumor suppressors. PTEN is an important tumor suppressor that is frequently mutated or deleted in human cancer. Loss of PTEN has been associated with aneuploidy and poor prognosis in cancer patients. In mice, Pten deletion or mutation drives genomic instability and tumor development. PTEN deficiency induces DNA replication stress, confers stress tolerance, and disrupts mitotic spindle architecture, leading to accumulation of structural and numerical chromosome instability. Therefore, PTEN guards the genome by controlling multiple processes of chromosome inheritance. Here, we summarize current understanding of the PTEN function in promoting high-fidelity transmission of genetic information. We also discuss the PTEN pathways of genome maintenance and highlight potential targets for cancer treatment. © 2017 WILEY Periodicals, Inc.

Dormant origins as a built-in safeguard in eukaryotic DNA replication against genome instability and disease development.

PubMed

Shima, Naoko; Pederson, Kayla D

2017-08-01

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

MYC and the Control of DNA Replication

PubMed Central

Dominguez-Sola, David; Gautier, Jean

2014-01-01

The MYC oncogene is a multifunctional protein that is aberrantly expressed in a significant fraction of tumors from diverse tissue origins. Because of its multifunctional nature, it has been difficult to delineate the exact contributions of MYC’s diverse roles to tumorigenesis. Here, we review the normal role of MYC in regulating DNA replication as well as its ability to generate DNA replication stress when overexpressed. Finally, we discuss the possible mechanisms by which replication stress induced by aberrant MYC expression could contribute to genomic instability and cancer. PMID:24890833

Importance of DNA repair in tumor suppression

NASA Astrophysics Data System (ADS)

Brumer, Yisroel; Shakhnovich, Eugene I.

2004-12-01

The transition from a normal to cancerous cell requires a number of highly specific mutations that affect cell cycle regulation, apoptosis, differentiation, and many other cell functions. One hallmark of cancerous genomes is genomic instability, with mutation rates far greater than those of normal cells. In microsatellite instability (MIN tumors), these are often caused by damage to mismatch repair genes, allowing further mutation of the genome and tumor progression. These mutation rates may lie near the error catastrophe found in the quasispecies model of adaptive RNA genomes, suggesting that further increasing mutation rates will destroy cancerous genomes. However, recent results have demonstrated that DNA genomes exhibit an error threshold at mutation rates far lower than their conservative counterparts. Furthermore, while the maximum viable mutation rate in conservative systems increases indefinitely with increasing master sequence fitness, the semiconservative threshold plateaus at a relatively low value. This implies a paradox, wherein inaccessible mutation rates are found in viable tumor cells. In this paper, we address this paradox, demonstrating an isomorphism between the conservatively replicating (RNA) quasispecies model and the semiconservative (DNA) model with post-methylation DNA repair mechanisms impaired. Thus, as DNA repair becomes inactivated, the maximum viable mutation rate increases smoothly to that of a conservatively replicating system on a transformed landscape, with an upper bound that is dependent on replication rates. On a specific single fitness peak landscape, the repair-free semiconservative system is shown to mimic a conservative system exactly. We postulate that inactivation of post-methylation repair mechanisms is fundamental to the progression of a tumor cell and hence these mechanisms act as a method for the prevention and destruction of cancerous genomes.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing.

PubMed

Laun, Peter; Bruschi, Carlo V; Dickinson, J Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing

PubMed Central

Laun, Peter; Bruschi, Carlo V.; Dickinson, J. Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast. PMID:17986449

The binding efficiency of RPA to telomeric G-strands folded into contiguous G-quadruplexes is independent of the number of G4 units.

PubMed

Lancrey, Astrid; Safa, Layal; Chatain, Jean; Delagoutte, Emmanuelle; Riou, Jean-François; Alberti, Patrizia; Saintomé, Carole

2018-03-01

Replication protein A (RPA) is a single-stranded DNA binding protein involved in replication and in telomere maintenance. During telomere replication, G-quadruplexes (G4) can accumulate on the lagging strand template and need to be resolved. It has been shown that human RPA is able to unfold a single G4. Nevertheless, the G-strand of human telomeres is prone to fold into higher-order structures formed by contiguous G-quadruplexes. To understand how RPA deals with these structures, we studied its interaction with telomeric G-strands folding into an increasing number of contiguous G4s. The aim of this study was to determine whether the efficiency of binding/unfolding of hRPA to telomeric G-strands depends on the number of G4 units. Our data show that the number n of contiguous G4 units (n ≥ 2) does not affect the efficiency of hRPA to coat transiently exposed single-stranded telomeric G-strands. This feature may be essential in preventing instability due to G4 structures during telomere replication. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Human RECQL5: guarding the crossroads of DNA replication and transcription and providing backup capability.

PubMed

Popuri, Venkateswarlu; Tadokoro, Takashi; Croteau, Deborah L; Bohr, Vilhelm A

2013-01-01

DNA helicases are ubiquitous enzymes that catalyze unwinding of duplex DNA and function in all metabolic processes in which access to single-stranded DNA is required, including DNA replication, repair, recombination and RNA transcription. RecQ helicases are a conserved family of DNA helicases that display highly specialized and vital roles in the maintenance of genome stability. Mutations in three of the five human RecQ helicases, BLM, WRN and RECQL4 are associated with the genetic disorders Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome that are characterized by chromosomal instability, premature aging and predisposition to cancer. The biological role of human RECQL5 is only partially understood and RECQL5 has not yet been associated with any human disease. Illegitimate recombination and replication stress are hallmarks of human cancers and common instigators for genomic instability and cell death. Recql5 knockout mice are cancer prone and show increased chromosomal instability. Recql5-deficient mouse embryonic fibroblasts are sensitive to camptothecin and display elevated levels of sister chromatid exchanges. Unlike other human RecQ helicases, RECQL5 is recruited to single-stranded DNA breaks and is also proposed to play an essential role in RNA transcription. Here, we review the established roles of RECQL5 at the cross roads of DNA replication, recombination and transcription, and propose that human RECQL5 provides important backup functions in the absence of other DNA helicases.

TGFβ signaling supports survival and metastasis of endometrial cancer cells

PubMed Central

Lei, XiuFen; Wang, Long; Yang, Junhua; Sun, Lu-Zhe

2009-01-01

The association of mutation of the transforming growth factor beta (TGFβ) type II receptor (RII) with microsatellite instability revealed a significant molecular mechanism of tumorigenesis and tumor progression in gastrointestinal carcinomas with DNA replication error. However, mutation of RII is rare in other types of carcinomas with microsatellite instability including endometrial adenocarcinoma suggesting that TGFβ receptor signaling may be necessary for tumor progression. To test this hypothesis, we abrogated TGFβ signaling with ectopic expression of a dominant-negative RII (DNRII) in human endometrial carcinoma HEC-1-A cells with microsatellite instability. Our study showed that over-expression of DNRII blocked the TGFβ signaling, inhibited anchorage-dependent and -independent growth, and stimulated apoptosis in vitro. Interestingly, the expression of DNRII expression showed little effect on tumor growth of subcutaneously inoculated cells in vivo. On the other hand, the DNRII cells showed more epithelial features whereas the control cells showed more mesenchymal features suggesting a reversal of autocrine TGFβ-induced epithelial–mesenchymal transition (EMT). Consistent with these findings, DNRII cells were much less migratory and invasive in vitro and metastatic in vivo than the control cells. Therefore, an intact TGFβ signaling pathway appears necessary for the metastatic phenotypes of this carcinoma model. PMID:20622970

Nonstandard Work and Marital Instability: Evidence from the National Longitudinal Survey of Youth

ERIC Educational Resources Information Center

Kalil, Ariel; Ziol-Guest, Kathleen M.; Epstein, Jodie Levin

2010-01-01

This article replicated and extended Harriet Presser's (2000) investigation of the linkages between nonstandard work and marital instability. We reexplored this question using data from a sample of 2,893 newlywed couples from the National Longitudinal Survey of Youth (NLSY) and using different analytic techniques. In contrast to Presser, we found…

DNA Secondary Structure at Chromosomal Fragile Sites in Human Disease

PubMed Central

Thys, Ryan G; Lehman, Christine E; Pierce, Levi C. T; Wang, Yuh-Hwa

2015-01-01

DNA has the ability to form a variety of secondary structures that can interfere with normal cellular processes, and many of these structures have been associated with neurological diseases and cancer. Secondary structure-forming sequences are often found at chromosomal fragile sites, which are hotspots for sister chromatid exchange, chromosomal translocations, and deletions. Structures formed at fragile sites can lead to instability by disrupting normal cellular processes such as DNA replication and transcription. The instability caused by disruption of replication and transcription can lead to DNA breakage, resulting in gene rearrangements and deletions that cause disease. In this review, we discuss the role of DNA secondary structure at fragile sites in human disease. PMID:25937814

Expression levels of DNA replication and repair genes predict regional somatic repeat instability in the brain but are not altered by polyglutamine disease protein expression or age.

PubMed

Mason, Amanda G; Tomé, Stephanie; Simard, Jodie P; Libby, Randell T; Bammler, Theodor K; Beyer, Richard P; Morton, A Jennifer; Pearson, Christopher E; La Spada, Albert R

2014-03-15

Expansion of CAG/CTG trinucleotide repeats causes numerous inherited neurological disorders, including Huntington's disease (HD), several spinocerebellar ataxias and myotonic dystrophy type 1. Expanded repeats are genetically unstable with a propensity to further expand when transmitted from parents to offspring. For many alleles with expanded repeats, extensive somatic mosaicism has been documented. For CAG repeat diseases, dramatic instability has been documented in the striatum, with larger expansions noted with advancing age. In contrast, only modest instability occurs in the cerebellum. Using microarray expression analysis, we sought to identify the genetic basis of these regional instability differences by comparing gene expression in the striatum and cerebellum of aged wild-type C57BL/6J mice. We identified eight candidate genes enriched in cerebellum, and validated four--Pcna, Rpa1, Msh6 and Fen1--along with a highly associated interactor, Lig1. We also explored whether expression levels of mismatch repair (MMR) proteins are altered in a line of HD transgenic mice, R6/2, that is known to show pronounced regional repeat instability. Compared with wild-type littermates, MMR expression levels were not significantly altered in R6/2 mice regardless of age. Interestingly, expression levels of these candidates were significantly increased in the cerebellum of control and HD human samples in comparison to striatum. Together, our data suggest that elevated expression levels of DNA replication and repair proteins in cerebellum may act as a safeguard against repeat instability, and may account for the dramatically reduced somatic instability present in this brain region, compared with the marked instability observed in the striatum.

Longitudinal Associations Between Marital Instability and Child Sleep Problems across Infancy and Toddlerhood in Adoptive Families

PubMed Central

Mannering, Anne M.; Harold, Gordon T.; Leve, Leslie D.; Shelton, Katherine H.; Shaw, Daniel S.; Conger, Rand D.; Neiderhiser, Jenae M.; Scaramella, Laura V.; Reiss, David

2009-01-01

This study examined the longitudinal association between marital instability and child sleep problems at ages 9 and 18 months in 357 families with a genetically unrelated infant adopted at birth. This design eliminates shared genes as an explanation for similarities between parent and child. Structural equation modeling indicated that T1 marital instability predicted T2 child sleep problems, but T1 child sleep problems did not predict T2 marital instability. This pattern of results was replicated when models were estimated separately for mothers and children and for fathers and children. Thus, even after controlling for stability in sleep problems and marital instability and eliminating shared genetic influences on associations using a longitudinal adoption design, marital instability prospectively predicts early childhood sleep patterns. PMID:21557740

Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance

PubMed Central

Li, Wenzhu; Yi, Jia; Agbu, Pamela; Zhou, Zheng; Kelley, Richard L.; Jia, Songtao

2017-01-01

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome. PMID:28749973

Mechanisms of DNA replication termination.

PubMed

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.

Child Care Instability and Maternal Depressive Symptoms: Exploring New Avenues for Supporting Maternal Mental Health.

PubMed

Johnson, Anna D; Padilla, Christina M

2018-05-28

We investigate links between child-care experiences - specifically care instability and mother's perceptions of care access - and maternal depressive symptoms, in an effort to illuminate policy-amenable mechanisms through which child-care experiences can support maternal mental health. Data come from the nationally representative Early Childhood Longitudinal Study - Birth Cohort (ECLS-B). We use regression models with lagged dependent variables to estimate associations between aspects of child-care instability and perceptions of care availability, and maternal depressive symptoms. We do so on the full sample and then on subgroups of mothers for whom child-care instability may be especially distressing: mothers who are low-income, working, single, or non-native speakers of English. Child-care instability - length in months in the longest arrangement, and number of arrangements - was not associated with maternal depressive symptoms. However, mothers' perception of having good choices for care was associated with a reduced likelihood of clinical depressive symptomology, even after controlling for prior depressive symptoms and concurrent parenting stress; this latter association was observed both in the full sample (Adjusted Odds Ratio [AOR]=0.77; 95% Confidence Interval [CI]=0.63-0.96) and among subgroups of employed mothers (AOR=0.71; CI=0.57-0.87) and single mothers (AOR=0.72; CI=0.52-0.99). Although dimensions of care instability did not associate with maternal depressive symptoms, mothers' perceptions of available care options, did. If replicated, findings would highlight a previously unconsidered avenue - increasing care accessibility and awareness of available options - for promoting maternal mental health in a population likely to experience depression but unlikely to be treated. Copyright © 2018. Published by Elsevier Inc.

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

PubMed

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.

Replication Stress: A Lifetime of Epigenetic Change

PubMed Central

Khurana, Simran; Oberdoerffer, Philipp

2015-01-01

DNA replication is essential for cell division. Challenges to the progression of DNA polymerase can result in replication stress, promoting the stalling and ultimately collapse of replication forks. The latter involves the formation of DNA double-strand breaks (DSBs) and has been linked to both genome instability and irreversible cell cycle arrest (senescence). Recent technological advances have elucidated many of the factors that contribute to the sensing and repair of stalled or broken replication forks. In addition to bona fide repair factors, these efforts highlight a range of chromatin-associated changes at and near sites of replication stress, suggesting defects in epigenome maintenance as a potential outcome of aberrant DNA replication. Here, we will summarize recent insight into replication stress-induced chromatin-reorganization and will speculate on possible adverse effects for gene expression, nuclear integrity and, ultimately, cell function. PMID:26378584

The Fanconi anemia pathway and ICL repair: implications for cancer therapy

PubMed Central

Wang, Lily C; Gautier, Jean

2011-01-01

Fanconi anemia (FA) is an inherited disease caused by mutations in at least 13 genes and characterized by genomic instability. In addition to displaying strikingly heterogenous clinical phenotypes, FA patients are exquisitely sensitive to treatments with crosslinking agents that create interstrand crosslinks (ICL). In contrast to bacteria and yeast, in which ICLs are repaired through replication-dependent and –independent mechanisms, it is thought that ICLs are repaired primarily during DNA replication in vertebrates (Moldovan and D’Andrea, 2009). However, recent data indicate that replication-independent ICL repair also operates in vertebrates. While the precise role of the FA pathway in ICL repair remains elusive, increasing evidence suggests that FA proteins function at different steps in the sensing, recognition and processing of ICLs, as well as in signaling from these very toxic lesions, which can be generated by a wide variety of cancer chemotherapeutic drugs. Here, we discuss some of the recent findings that have shed light on the role of the FA pathway in ICL repair with special emphasis on the implications of these findings for cancer therapy since disruption of FA genes have been associated with cancer predisposition. PMID:20807115

Longitudinal associations between marital instability and child sleep problems across infancy and toddlerhood in adoptive families.

PubMed

Mannering, Anne M; Harold, Gordon T; Leve, Leslie D; Shelton, Katherine H; Shaw, Daniel S; Conger, Rand D; Neiderhiser, Jenae M; Scaramella, Laura V; Reiss, David

2011-01-01

This study examined the longitudinal association between marital instability and child sleep problems at ages 9 and 18 months in 357 families with a genetically unrelated infant adopted at birth. This design eliminates shared genes as an explanation for similarities between parent and child. Structural equation modeling indicated that T1 marital instability predicted T2 child sleep problems, but T1 child sleep problems did not predict T2 marital instability. This result was replicated when models were estimated separately for mothers and fathers. Thus, even after controlling for stability in sleep problems and marital instability and eliminating shared genetic influences on associations using a longitudinal adoption design, marital instability prospectively predicts early childhood sleep patterns. © 2011 The Authors. Child Development © 2011 Society for Research in Child Development, Inc.

FANCJ suppresses microsatellite instability and lymphomagenesis independent of the Fanconi anemia pathway.

PubMed

Matsuzaki, Kenichiro; Borel, Valerie; Adelman, Carrie A; Schindler, Detlev; Boulton, Simon J

2015-12-15

Microsatellites are short tandem repeat sequences that are highly prone to expansion/contraction due to their propensity to form non-B-form DNA structures, which hinder DNA polymerases and provoke template slippage. Although error correction by mismatch repair plays a key role in preventing microsatellite instability (MSI), which is a hallmark of Lynch syndrome, activities must also exist that unwind secondary structures to facilitate replication fidelity. Here, we report that Fancj helicase-deficient mice, while phenotypically resembling Fanconi anemia (FA), are also hypersensitive to replication inhibitors and predisposed to lymphoma. Whereas metabolism of G4-DNA structures is largely unaffected in Fancj(-/-) mice, high levels of spontaneous MSI occur, which is exacerbated by replication inhibition. In contrast, MSI is not observed in Fancd2(-/-) mice but is prevalent in human FA-J patients. Together, these data implicate FANCJ as a key factor required to counteract MSI, which is functionally distinct from its role in the FA pathway. © 2015 Matsuzaki et al.; Published by Cold Spring Harbor Laboratory Press.

A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

PubMed

Kabeche, Lilian; Nguyen, Hai Dang; Buisson, Rémi; Zou, Lee

2018-01-05

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability. Copyright © 2018, American Association for the Advancement of Science.

Assembly of Slx4 signaling complexes behind DNA replication forks.

PubMed

Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

2015-08-13

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. © 2015 The Authors.

The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities.

PubMed

Naim, Valeria; Rosselli, Filippo

2009-06-01

Loss-of-function of caretaker genes characterizes a group of cancer predisposition diseases that feature cellular hypersensitivity to DNA damage and chromosome fragility; this group includes Fanconi anaemia and Bloom syndrome. The products of the 13 FANC genes (mutated in Fanconi anaemia), which constitute the 'FANC' pathway, and BLM (the RecQ helicase mutated in Bloom syndrome) are thought to collaborate during the S phase of the cell cycle, preventing chromosome instability. Recently, BLM has been implicated in the completion of sister chromatid separation during mitosis, a complex process in which precise regulation and execution is crucial to preserve genomic stability. Here we show for the first time a role for the FANC pathway in chromosome segregation during mitotic cell division. FANCD2, a key component of the pathway, localizes to discrete spots on mitotic chromosomes. FANCD2 chromosomal localization is responsive to replicative stress and specifically targets aphidicolin (APH)-induced chromatid gaps and breaks. Our data indicate that the FANC pathway is involved in rescuing abnormal anaphase and telophase (ana-telophase) cells, limiting aneuploidy and reducing chromosome instability in daughter cells. We further address a cooperative role for the FANC pathway and BLM in preventing micronucleation, through FANC-dependent targeting of BLM to non-centromeric abnormal structures induced by replicative stress. We reveal new crosstalk between FANC and BLM proteins, extending their interaction beyond the S-phase rescue of damaged DNA to the safeguarding of chromosome stability during mitosis.

Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline

PubMed Central

Mendez-Bermudez, Aaron; Hills, Mark; Pickett, Hilda A.; Phan, Anh Tuân; Mergny, Jean-Louis; Riou, Jean-François; Royle, Nicola J.

2009-01-01

A number of different processes that impact on telomere length dynamics have been identified but factors that affect the turnover of repeats located proximally within the telomeric DNA are poorly defined. We have identified a particular repeat type (CTAGGG) that is associated with an extraordinarily high mutation rate (20% per gamete) in the male germline. The mutation rate is affected by the length and sequence homogeneity of the (CTAGGG)n array. This level of instability was not seen with other sequence-variant repeats, including the TCAGGG repeat type that has the same composition. Telomeres carrying a (CTAGGG)n array are also highly unstable in somatic cells with the mutation process resulting in small gains or losses of repeats that also occasionally result in the deletion of the whole (CTAGGG)n array. These sequences are prone to quadruplex formation in vitro but adopt a different topology from (TTAGGG)n (see accompanying article). Interestingly, short (CTAGGG)2 oligonucleotides induce a DNA damage response (γH2AX foci) as efficiently as (TTAGGG)2 oligos in normal fibroblast cells, suggesting they recruit POT1 from the telomere. Moreover, in vitro assays show that (CTAGGG)n repeats bind POT1 more efficiently than (TTAGGG)n or (TCAGGG)n. We estimate that 7% of human telomeres contain (CTAGGG)n repeats and when present, they create additional problems that probably arise during telomere replication. PMID:19656953

Unstable genomes elevate transcriptome dynamics

PubMed Central

Stevens, Joshua B.; Liu, Guo; Abdallah, Batoul Y.; Horne, Steven D.; Ye, Karen J.; Bremer, Steven W.; Ye, Christine J.; Krawetz, Stephen A.; Heng, Henry H.

2015-01-01

The challenge of identifying common expression signatures in cancer is well known, however the reason behind this is largely unclear. Traditionally variation in expression signatures has been attributed to technological problems, however recent evidence suggests that chromosome instability (CIN) and resultant karyotypic heterogeneity may be a large contributing factor. Using a well-defined model of immortalization, we systematically compared the pattern of genome alteration and expression dynamics during somatic evolution. Co-measurement of global gene expression and karyotypic alteration throughout the immortalization process reveals that karyotype changes influence gene expression as major structural and numerical karyotypic alterations result in large gene expression deviation. Replicate samples from stages with stable genomes are more similar to each other than are replicate samples with karyotypic heterogeneity. Karyotypic and gene expression change during immortalization is dynamic as each stage of progression has a unique expression pattern. This was further verified by comparing global expression in two replicates grown in one flask with known karyotypes. Replicates with higher karyotypic instability were found to be less similar than replicates with stable karyotypes. This data illustrates the karyotype, transcriptome, and transcriptome determined pathways are in constant flux during somatic cellular evolution (particularly during the macroevolutionary phase) and this flux is an inextricable feature of CIN and essential for cancer formation. The findings presented here underscore the importance of understanding the evolutionary process of cancer in order to design improved treatment modalities. PMID:24122714

Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

PubMed

Patterson, Melissa N; Maxwell, Patrick H

2014-10-16

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Cancer therapy and replication stress: forks on the road to perdition.

PubMed

Kotsantis, Panagiotis; Jones, Rebecca M; Higgs, Martin R; Petermann, Eva

2015-01-01

Deregulated DNA replication occurs in cancer where it contributes to genomic instability. This process is a target of cytotoxic therapies. Chemotherapies exploit high DNA replication in cancer cells by modifying the DNA template or by inhibiting vital enzymatic activities that lead to slowing or stalling replication fork progression. Stalled replication forks can be converted into toxic DNA double-strand breaks resulting in cell death, i.e., replication stress. While likely crucial for many cancer treatments, replication stress is poorly understood due to its complexity. While we still know relatively little about the role of replication stress in cancer therapy, technical advances in recent years have shed new light on the effect that cancer therapeutics have on replication forks and the molecular mechanisms that lead from obstructed fork progression to cell death. This chapter will give an overview of our current understanding of replication stress in the context of cancer therapy. © 2015 Elsevier Inc. All rights reserved.

Mitochondria damage checkpoint in apoptosis and genome stability.

PubMed

Singh, Keshav K

2004-11-01

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

An H2A Histone Isotype, H2ac, Associates with Telomere and Maintains Telomere Integrity

PubMed Central

Tzeng, Tsai-Yu; Lin, I-Hsuan; Hsu, Ming-Ta

2016-01-01

Telomeres are capped at the ends of eukaryotic chromosomes and are composed of TTAGGG repeats bound to the shelterin complex. Here we report that a replication-dependent histone H2A isotype, H2ac, was associated with telomeres in human cells and co-immunoprecipitates with telomere repeat factor 2 (TRF2) and protection of telomeres protein 1 (POT1), whereas other histone H2A isotypes and mutations of H2ac did not bind to telomeres or these two proteins. The amino terminal basic domain of TRF2 was necessary for the association with H2ac and for the recruitment of H2ac to telomeres. Depletion of H2ac led to loss of telomeric repeat sequences, the appearance of dysfunctional telomeres, and chromosomal instability, including chromosomal breaks and anaphase bridges, as well as accumulation of telomere-associated DNA damage factors in H2ac depleted cells. Additionally, knockdown of H2ac elicits an ATM-dependent DNA damage response at telomeres and depletion of XPF protects telomeres against H2ac-deficiency-induced G-strand overhangs loss and DNA damage response, and prevents chromosomal instability. These findings suggest that the H2A isotype, H2ac, plays an essential role in maintaining telomere functional integrity. PMID:27228173

Adventures in hepatocarcinogenesis.

PubMed

Pitot, Henry C

2007-01-01

Neoplasia is a heritably altered, relatively autonomous growth of tissue. Hepatocarcinogenesis, the pathogenesis of neoplasia in liver, as modeled in the rat exhibits three distinct, quantifiable stages: initiation, promotion, and progression. Simple mutations and/or epigenetic alterations may result in the irreversible stage of initiation. The stage of promotion results from selective enhancement of cell replication and selective inhibition of cellular apoptosis of initiated cells dependent on the genetic and/or epigenetic alterations of the latter. The irreversible stage of progression results from initial karyotypic alterations that evolve into greater degrees of genomic instability. The initial genomic alteration in the transition from promotion to progression may involve primarily epigenetic mechanisms driven by epigenetic and genetic alterations fixed during the stage of promotion.

A distinct first replication cycle of DNA introduced in mammalian cells

PubMed Central

Chandok, Gurangad S.; Kapoor, Kalvin K.; Brick, Rachel M.; Sidorova, Julia M.; Krasilnikova, Maria M.

2011-01-01

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells. PMID:21062817

Engine-Scale Combustor Rig Designed, Fabricated, and Tested for Combustion Instability Control Research

NASA Technical Reports Server (NTRS)

DeLaat, John C.; Breisacher, Kevin J.

2000-01-01

Low-emission combustor designs are prone to combustor instabilities. Because active control of these instabilities may allow future combustors to meet both stringent emissions and performance requirements, an experimental combustor rig was developed for investigating methods of actively suppressing combustion instabilities. The experimental rig has features similar to a real engine combustor and exhibits instabilities representative of those in aircraft gas turbine engines. Experimental testing in the spring of 1999 demonstrated that the rig can be tuned to closely represent an instability observed in engine tests. Future plans are to develop and demonstrate combustion instability control using this experimental combustor rig. The NASA Glenn Research Center at Lewis Field is leading the Combustion Instability Control program to investigate methods for actively suppressing combustion instabilities. Under this program, a single-nozzle, liquid-fueled research combustor rig was designed, fabricated, and tested. The rig has many of the complexities of a real engine combustor, including an actual fuel nozzle and swirler, dilution cooling, and an effusion-cooled liner. Prior to designing the experimental rig, a survey of aircraft engine combustion instability experience identified an instability observed in a prototype engine as a suitable candidate for replication. The frequency of the instability was 525 Hz, with an amplitude of approximately 1.5-psi peak-to-peak at a burner pressure of 200 psia. The single-nozzle experimental combustor rig was designed to preserve subcomponent lengths, cross sectional area distribution, flow distribution, pressure-drop distribution, temperature distribution, and other factors previously found to be determinants of burner acoustic frequencies, mode shapes, gain, and damping. Analytical models were used to predict the acoustic resonances of both the engine combustor and proposed experiment. The analysis confirmed that the test rig configuration and engine configuration had similar longitudinal acoustic characteristics, increasing the likelihood that the engine instability would be replicated in the rig. Parametric analytical studies were performed to understand the influence of geometry and condition variations and to establish a combustion test plan. Cold-flow experiments verified that the design values of area and flow distributions were obtained. Combustion test results established the existence of a longitudinal combustion instability in the 500-Hz range with a measured amplitude approximating that observed in the engine. Modifications to the rig configuration during testing also showed the potential for injector independence. The research combustor rig was developed in partnership with Pratt & Whitney of West Palm Beach, Florida, and United Technologies Research Center of East Hartford, Connecticut. Experimental testing of the combustor rig took place at United Technologies Research Center.

DNA replication stress and cancer: cause or cure?

PubMed

Taylor, Elaine M; Lindsay, Howard D

2016-01-01

There is an extensive and growing body of evidence that DNA replication stress is a major driver in the development and progression of many cancers, and that these cancers rely heavily on replication stress response pathways for their continued proliferation. This raises the possibility that the pathways that ordinarily protect cells from the accumulation of cancer-causing mutations may actually prove to be effective therapeutic targets for a wide range of malignancies. In this review, we explore the mechanisms by which sustained proliferation can lead to replication stress and genome instability, and discuss how the pattern of mutations observed in human cancers is supportive of this oncogene-induced replication stress model. Finally, we go on to consider the implications of replication stress both as a prognostic indicator and, more encouragingly, as a potential target in cancer treatment.

Physical proximity of chromatin to nuclear pores prevents harmful R loop accumulation contributing to maintain genome stability.

PubMed

García-Benítez, Francisco; Gaillard, Hélène; Aguilera, Andrés

2017-10-10

During transcription, the mRNA may hybridize with DNA, forming an R loop, which can be physiological or pathological, constituting in this case a source of genomic instability. To understand the mechanism by which eukaryotic cells prevent harmful R loops, we used human activation-induced cytidine deaminase (AID) to identify genes preventing R loops. A screening of 400 Saccharomyces cerevisiae selected strains deleted in nuclear genes revealed that cells lacking the Mlp1/2 nuclear basket proteins show AID-dependent genomic instability and replication defects that were suppressed by RNase H1 overexpression. Importantly, DNA-RNA hybrids accumulated at transcribed genes in mlp1/2 mutants, indicating that Mlp1/2 prevents R loops. Consistent with the Mlp1/2 role in gene gating to nuclear pores, artificial tethering to the nuclear periphery of a transcribed locus suppressed R loops in mlp1 ∆ cells. The same occurred in THO-deficient hpr1 ∆ cells. We conclude that proximity of transcribed chromatin to the nuclear pore helps restrain pathological R loops.

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

PubMed

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.

Reproductive potential and instability of the rDNA region of the Saccharomyces cerevisiae yeast: Common or separate mechanisms of regulation?

PubMed

Zadrag-Tecza, Renata; Skoneczna, Adrianna

2016-11-01

The yeast Saccharomyces cerevisiae is a unicellular organism commonly used as a model to explain mechanisms of aging in multicellular organisms. It is used as a model organism for both replicative and chronological aging. Replicative aging is defined as the number of daughter cells produced by an individual cell during its life. A widely accepted hypothesis assumes that replicative aging of yeast is related to the existence of a so called "senescence factor" that gradually accumulates in the mother cell, which consequently leads to its death. One of the earliest proposed "senescence factors" were extrachromosomal rDNA circles (ERCs). However, their role in the regulation of the replicative lifespan is somewhat controversial and subject to discussion. In this paper, we propose a more comprehensive approach to this problem by analysing the length of life and the correlation between the cell size and the replicative lifespan of yeast cells with different level of ERCs, i.e. Δrad52 and Δsgs1 mutants. This analysis shows that it is not the accumulation of ERCs but genomic instability and hypertrophy that play an important role in the regulation of reproductive potential and total lifespan of the S. cerevisiae yeast. However, these two factors have a different impact on various phases of the yeast cell life, i.e. reproductive and post-reproductive phases. Copyright © 2016 Elsevier Inc. All rights reserved.

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

PubMed

Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

2016-09-01

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase*

PubMed Central

Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.

2014-01-01

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.

PubMed

Zhu, Quan; Hoong, Nien; Aslanian, Aaron; Hara, Toshiro; Benner, Christopher; Heinz, Sven; Miga, Karen H; Ke, Eugene; Verma, Sachin; Soroczynski, Jan; Yates, John R; Hunter, Tony; Verma, Inder M

2018-06-07

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Analysis of re-replication from deregulated origin licensing by DNA fiber spreading

PubMed Central

Dorn, Elizabeth S.; Chastain, Paul D.; Hall, Jonathan R.; Cook, Jeanette Gowen

2009-01-01

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability. PMID:19010964

Fanconi anemia proteins in telomere maintenance.

PubMed

Sarkar, Jaya; Liu, Yie

2016-07-01

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell. Published by Elsevier B.V.

Back to the Origin

PubMed Central

Evertts, Adam G.

2012-01-01

In bacteria, replication is a carefully orchestrated event that unfolds the same way for each bacterium and each cell division. The process of DNA replication in bacteria optimizes cell growth and coordinates high levels of simultaneous replication and transcription. In metazoans, the organization of replication is more enigmatic. The lack of a specific sequence that defines origins of replication has, until recently, severely limited our ability to define the organizing principles of DNA replication. This question is of particular importance as emerging data suggest that replication stress is an important contributor to inherited genetic damage and the genomic instability in tumors. We consider here the replication program in several different organisms including recent genome-wide analyses of replication origins in humans. We review recent studies on the role of cytosine methylation in replication origins, the role of transcriptional looping and gene gating in DNA replication, and the role of chromatin’s 3-dimensional structure in DNA replication. We use these new findings to consider several questions surrounding DNA replication in metazoans: How are origins selected? What is the relationship between replication and transcription? How do checkpoints inhibit origin firing? Why are there early and late firing origins? We then discuss whether oncogenes promote cancer through a role in DNA replication and whether errors in DNA replication are important contributors to the genomic alterations and gene fusion events observed in cancer. We conclude with some important areas for future experimentation. PMID:23634256

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

PubMed

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.

Two-stream instability with time-dependent drift velocity

DOE PAGES

Qin, Hong; Davidson, Ronald C.

2014-06-26

The classical two-stream instability driven by a constant relative drift velocity between two plasma components is extended to the case with time-dependent drift velocity. A solution method is developed to rigorously define and calculate the instability growth rate for linear perturbations relative to the time-dependent unperturbed two-stream motions. The stability diagrams for the oscillating two-stream instability are presented over a large region of parameter space. It is shown that the growth rate for the classical two-stream instability can be significantly reduced by adding an oscillatory component to the relative drift velocity.

Phosphorylation by CK2 regulates MUS81/EME1 in mitosis and after replication stress.

PubMed

Palma, Anita; Pugliese, Giusj Monia; Murfuni, Ivana; Marabitti, Veronica; Malacaria, Eva; Rinalducci, Sara; Minoprio, Anna; Sanchez, Massimo; Mazzei, Filomena; Zolla, Lello; Franchitto, Annapaola; Pichierri, Pietro

2018-06-01

The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis.

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

PubMed

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.

Measuring the Levels of Ribonucleotides Embedded in Genomic DNA.

PubMed

Meroni, Alice; Nava, Giulia M; Sertic, Sarah; Plevani, Paolo; Muzi-Falconi, Marco; Lazzaro, Federico

2018-01-01

Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.

HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4.

PubMed

Das, Mitali; Prasad, Shyam Babu; Yadav, Suresh Singh; Modi, Arusha; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

2015-12-01

Minichoromosome maintenance (MCM) proteins play key role in cell cycle progression by licensing DNA replication only once per cell cycle. These proteins are found to be overexpressed in cervical cancer cells. In this study, we depleted MCM4, one of the MCM 2-7 complex components by RNA interference (RNAi) in four cervical cancer cell lines. The four cell lines were selected on the basis of their human papillomavirus (HPV) infection: HPV16-positive SiHa, HPV18-positive ME-180, HPV16- and HPV18-positive CaSki, and HPV-negative C-33A. The MCM4-deficient cells irrespective of their HPV status grow for several generations and maintain regular cell cycle. We did not find any evidence of augmented response to a short-term (48 h) cisplatin treatment in these MCM4-deficient cells. However, MCM4-/HPV16+ SiHa cells cannot withstand a prolonged treatment (up to 5 days) of even a sublethal dosage of cisplatin. They show increased chromosomal instability compared to their control counterparts. On the other hand, MCM4-deficient CaSki cells (both HPV16+ and 18+) remain resistant to a prolonged exposure to cisplatin. Our study indicates that cervical cancer cells may be using excess MCMs as a backup for replicative stress; however, its regulatory mechanism is dependent on the HPV status of the cells.

Epigenetic Instability due to Defective Replication of Structured DNA

PubMed Central

Sarkies, Peter; Reams, Charlie; Simpson, Laura J.; Sale, Julian E.

2010-01-01

Summary The accurate propagation of histone marks during chromosomal replication is proposed to rely on the tight coupling of replication with the recycling of parental histones to the daughter strands. Here, we show in the avian cell line DT40 that REV1, a key regulator of DNA translesion synthesis at the replication fork, is required for the maintenance of repressive chromatin marks and gene silencing in the vicinity of DNA capable of forming G-quadruplex (G4) structures. We demonstrate a previously unappreciated requirement for REV1 in replication of G4 forming sequences and show that transplanting a G4 forming sequence into a silent locus leads to its derepression in REV1-deficient cells. Together, our observations support a model in which failure to maintain processive DNA replication at G4 DNA in REV1-deficient cells leads to uncoupling of DNA synthesis from histone recycling, resulting in localized loss of repressive chromatin through biased incorporation of newly synthesized histones. PMID:21145480

Nonenzymatic Role for WRN in Preserving Nascent DNA Strands after Replication Stress

DOE PAGES

Su, Fengtao; Mukherjee, Shibani; Yang, Yanyong; ...

2014-11-20

WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRNmore » to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.« less

Characterization and Simulation of the Thermoacoustic Instability Behavior of an Advanced, Low Emissions Combustor Prototype

NASA Technical Reports Server (NTRS)

DeLaat, John C.; Paxson, Daniel E.

2008-01-01

Extensive research is being done toward the development of ultra-low-emissions combustors for aircraft gas turbine engines. However, these combustors have an increased susceptibility to thermoacoustic instabilities. This type of instability was recently observed in an advanced, low emissions combustor prototype installed in a NASA Glenn Research Center test stand. The instability produces pressure oscillations that grow with increasing fuel/air ratio, preventing full power operation. The instability behavior makes the combustor a potentially useful test bed for research into active control methods for combustion instability suppression. The instability behavior was characterized by operating the combustor at various pressures, temperatures, and fuel and air flows representative of operation within an aircraft gas turbine engine. Trends in instability behavior versus operating condition have been identified and documented, and possible explanations for the trends provided. A simulation developed at NASA Glenn captures the observed instability behavior. The physics-based simulation includes the relevant physical features of the combustor and test rig, employs a Sectored 1-D approach, includes simplified reaction equations, and provides time-accurate results. A computationally efficient method is used for area transitions, which decreases run times and allows the simulation to be used for parametric studies, including control method investigations. Simulation results show that the simulation exhibits a self-starting, self-sustained combustion instability and also replicates the experimentally observed instability trends versus operating condition. Future plans are to use the simulation to investigate active control strategies to suppress combustion instabilities and then to experimentally demonstrate active instability suppression with the low emissions combustor prototype, enabling full power, stable operation.

Characterization and Simulation of Thermoacoustic Instability in a Low Emissions Combustor Prototype

NASA Technical Reports Server (NTRS)

DeLaat, John C.; Paxson, Daniel E.

2008-01-01

Extensive research is being done toward the development of ultra-low-emissions combustors for aircraft gas turbine engines. However, these combustors have an increased susceptibility to thermoacoustic instabilities. This type of instability was recently observed in an advanced, low emissions combustor prototype installed in a NASA Glenn Research Center test stand. The instability produces pressure oscillations that grow with increasing fuel/air ratio, preventing full power operation. The instability behavior makes the combustor a potentially useful test bed for research into active control methods for combustion instability suppression. The instability behavior was characterized by operating the combustor at various pressures, temperatures, and fuel and air flows representative of operation within an aircraft gas turbine engine. Trends in instability behavior vs. operating condition have been identified and documented. A simulation developed at NASA Glenn captures the observed instability behavior. The physics-based simulation includes the relevant physical features of the combustor and test rig, employs a Sectored 1-D approach, includes simplified reaction equations, and provides time-accurate results. A computationally efficient method is used for area transitions, which decreases run times and allows the simulation to be used for parametric studies, including control method investigations. Simulation results show that the simulation exhibits a self-starting, self-sustained combustion instability and also replicates the experimentally observed instability trends vs. operating condition. Future plans are to use the simulation to investigate active control strategies to suppress combustion instabilities and then to experimentally demonstrate active instability suppression with the low emissions combustor prototype, enabling full power, stable operation.

Effects of camptothecin or TOP1 overexpression on genetic stability in Saccharomyces cerevisiae.

PubMed

Sloan, Roketa; Huang, Shar-Yin Naomi; Pommier, Yves; Jinks-Robertson, Sue

2017-11-01

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation. Copyright © 2017 Elsevier B.V. All rights reserved.

Genes and Pathways Involved in Adult Onset Disorders Featuring Muscle Mitochondrial DNA Instability

PubMed Central

Ahmed, Naghia; Ronchi, Dario; Comi, Giacomo Pietro

2015-01-01

Replication and maintenance of mtDNA entirely relies on a set of proteins encoded by the nuclear genome, which include members of the core replicative machinery, proteins involved in the homeostasis of mitochondrial dNTPs pools or deputed to the control of mitochondrial dynamics and morphology. Mutations in their coding genes have been observed in familial and sporadic forms of pediatric and adult-onset clinical phenotypes featuring mtDNA instability. The list of defects involved in these disorders has recently expanded, including mutations in the exo-/endo-nuclease flap-processing proteins MGME1 and DNA2, supporting the notion that an enzymatic DNA repair system actively takes place in mitochondria. The results obtained in the last few years acknowledge the contribution of next-generation sequencing methods in the identification of new disease loci in small groups of patients and even single probands. Although heterogeneous, these genes can be conveniently classified according to the pathway to which they belong. The definition of the molecular and biochemical features of these pathways might be helpful for fundamental knowledge of these disorders, to accelerate genetic diagnosis of patients and the development of rational therapies. In this review, we discuss the molecular findings disclosed in adult patients with muscle pathology hallmarked by mtDNA instability. PMID:26251896

Genomic instability in cancer: Teetering on the limit of tolerance

PubMed Central

Andor, Noemi; Maley, Carlo C.; Ji, Hanlee P.

2017-01-01

Cancer genomic instability contributes to the phenomenon of intratumoral genetic heterogeneity, provides the genetic diversity required for natural selection and enables the extensive phenotypic diversity that is frequently observed among patients. Genomic instability has previously been associated with poor prognosis. However, we have evidence that for solid tumors of epithelial origin, extreme levels of genomic instability, where more than 75% of the genome is subject to somatic copy number alterations, are associated with a potentially better prognosis compared to intermediate levels under this threshold. This has been observed in clonal subpopulations of larger size, especially when genomic instability is shared among a limited number of clones. We hypothesize that cancers with extreme levels of genomic instability may be teetering on the brink of a threshold where so much of their genome is adversely altered that cells rarely replicate successfully. Another possibility is that tumors with high levels of genomic instability are more immunogenic than other cancers with a less extensive burden of genetic aberrations. Regardless of the exact mechanism, but hinging on our ability to quantify how a tumor’s burden of genetic aberrations is distributed among coexisting clones – genomic instability has important therapeutic implications. Herein, we explore the possibility that a high genomic instability could be the basis for a tumor’s sensitivity to DNA damaging therapies. We primarily focus on studies of epithelial-derived solid tumors. PMID:28432052

Zim17/Tim15 links mitochondrial iron-sulfur cluster biosynthesis to nuclear genome stability.

PubMed

Díaz de la Loza, María Del Carmen; Gallardo, Mercedes; García-Rubio, María Luisa; Izquierdo, Alicia; Herrero, Enrique; Aguilera, Andrés; Wellinger, Ralf Erik

2011-08-01

Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron-sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron-sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron-sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants.

Recent Insights into the Control of Human Papillomavirus (HPV) Genome Stability, Loss, and Degradation.

PubMed

Fisher, Chris

2015-01-01

Most human papillomavirus (HPV) antiviral strategies have focused upon inhibiting viral DNA replication, but it is increasingly apparent that viral DNA levels can be chemically controlled by approaches that promote its instability. HPVs and other DNA viruses have a tenuous relationship with their hosts. They must replicate and hide from the DNA damage response (DDR) and innate immune systems, which serve to protect cells from foreign or "non-self" DNA, and yet they draft these same systems to support their life cycles. DNA binding antiviral agents promoting massive viral DNA instability and elimination are reviewed. Mechanistic studies of these agents have identified genetic antiviral enhancers and repressors, antiviral sensitizers, and host cell elements that protect and stabilize HPV genomes. Viral DNA degradation appears to be an important means of controlling HPV DNA levels in some cases, but the underlying mechanisms remain poorly understood. These findings may prove useful not only for understanding viral DNA persistence but also in devising future antiviral strategies.

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

PubMed

Xue, Yuezhen; Toh, Shen Yon; He, Pingping; Lim, Thimothy; Lim, Diana; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

2015-10-27

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.

Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights

PubMed Central

Thompson, Larry H.; Hinz, John M.

2009-01-01

The Fanconi anemia (FA) molecular network consists of 15 “FANC” proteins, of which 13 are associated with mutations in patients with this cancer-prone chromosome instability disorder. Whereas historically the common phenotype associated with FA mutations is marked sensitivity to DNA interstrand crosslinking agents, the literature supports a more global role for FANC proteins in coping with diverse stresses encountered by replicative polymerases. We have attempted to reconcile and integrate numerous observations into a model in which FANC proteins coordinate the following physiological events during DNA crosslink repair: (a) activating a FANCM-ATR-dependent S-phase checkpoint; (b) mediating enzymatic replication-fork breakage and crosslink unhooking; (c) filling the resulting gap by translesion synthesis (TLS) by error-prone polymerase(s); and (d) restoring the resulting one-ended double-strand break by homologous recombination repair (HRR). The FANC core subcomplex (FANCA, B, C, E, F, G, L, FAAP100) promotes TLS for both crosslink and non-crosslink damage such as spontaneous oxidative base damage, UV-C photoproducts, and alkylated bases. TLS likely helps prevent stalled replication forks from breaking, thereby maintaining chromosome continuity. Diverse DNA damages and replication inhibitors result in monoubiquitination of the FANCD2-FANCI complex by the FANCL ubiquitin ligase activity of the core subcomplex upon its recruitment to chromatin by the FANCM-FAAP24 heterodimeric translocase. We speculate that this translocase activity acts as the primary damage sensor and helps remodel blocked replication forks to facilitate checkpoint activation and repair. Monoubiquitination of FANCD2-FANCI is needed for promoting HRR, in which the FANCD1/BRCA2 and FANCN/PALB2 proteins act at an early step. We conclude that the core subcomplex is required for both TLS and HRR occurring separately for non-crosslink damages and for both events during crosslink repair. The FANCJ/BRIP1/BACH1 helicase functions in association with BRCA1 and may remove structural barriers to replication, such as guanine quadruplex structures, and/or assist in crosslink unhooking. PMID:19622404

Demonstration of Active Combustion Control

NASA Technical Reports Server (NTRS)

Lovett, Jeffrey A.; Teerlinck, Karen A.; Cohen, Jeffrey M.

2008-01-01

The primary objective of this effort was to demonstrate active control of combustion instabilities in a direct-injection gas turbine combustor that accurately simulates engine operating conditions and reproduces an engine-type instability. This report documents the second phase of a two-phase effort. The first phase involved the analysis of an instability observed in a developmental aeroengine and the design of a single-nozzle test rig to replicate that phenomenon. This was successfully completed in 2001 and is documented in the Phase I report. This second phase was directed toward demonstration of active control strategies to mitigate this instability and thereby demonstrate the viability of active control for aircraft engine combustors. This involved development of high-speed actuator technology, testing and analysis of how the actuation system was integrated with the combustion system, control algorithm development, and demonstration testing in the single-nozzle test rig. A 30 percent reduction in the amplitude of the high-frequency (570 Hz) instability was achieved using actuation systems and control algorithms developed within this effort. Even larger reductions were shown with a low-frequency (270 Hz) instability. This represents a unique achievement in the development and practical demonstration of active combustion control systems for gas turbine applications.

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

PubMed

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.

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks*

PubMed Central

Kim, Hyun-Suk; Nickoloff, Jac A.; Wu, Yuehan; Williamson, Elizabeth A.; Sidhu, Gurjit Singh; Reinert, Brian L.; Jaiswal, Aruna S.; Srinivasan, Gayathri; Patel, Bhavita; Kong, Kimi; Burma, Sandeep; Lee, Suk-Hee; Hromas, Robert A.

2017-01-01

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks. PMID:28049724

DNA replication depends on photosynthetic electron transport in cyanobacteria.

PubMed

Ohbayashi, Ryudo; Watanabe, Satoru; Kanesaki, Yu; Narikawa, Rei; Chibazakura, Taku; Ikeuchi, Masahiko; Yoshikawa, Hirofumi

2013-07-01

The freshwater cyanobacterium Synechococcus elongatus PCC 7942 exhibits light-dependent growth. Although it has been reported that DNA replication also depends on light irradiation in S. elongatus 7942, the involvement of the light in the regulation of DNA replication remains unclear. To elucidate the regulatory pathway of DNA replication by light, we studied the effect of several inhibitors, including two electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), on DNA replication in S. elongatus 7942. DCMU inhibited only DNA replication initiation, whereas DBMIB blocked both the initiation and progression of DNA replication. These results suggest that DNA replication depends on the photosynthetic electron transport activity and initiation and progression of DNA replication are regulated in different ways. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Defects in Mitochondrial DNA Replication and Human Disease

PubMed Central

Copeland, William C.

2011-01-01

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes (MDS) such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease. PMID:22176657

Measuring DNA Replication in Hypoxic Conditions.

PubMed

Foskolou, Iosifina P; Biasoli, Deborah; Olcina, Monica M; Hammond, Ester M

2016-01-01

It is imperative that dividing cells maintain replication fork integrity in order to prevent DNA damage and cell death. The investigation of DNA replication is of high importance as alterations in this process can lead to genomic instability, a known causative factor of tumor development. A simple, sensitive, and informative technique which enables the study of DNA replication, is the DNA fiber assay, an adaptation of which is described in this chapter. The DNA fiber method is a powerful tool, which allows the quantitative and qualitative analysis of DNA replication at the single molecule level. The sequential pulse labeling of live cells with two thymidine analogues and the subsequent detection with specific antibodies and fluorescence imaging allows direct examination of sites of DNA synthesis. In this chapter, we describe how this assay can be performed in conditions of low oxygen levels (hypoxia)-a physiologically relevant stress that occurs in most solid tumors. Moreover, we suggest ways on how to overcome the technical problems that arise while using the hypoxic chambers.

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase.

PubMed

Takahashi, Shuntaro; Brazier, John A; Sugimoto, Naoki

2017-09-05

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

PubMed Central

Takahashi, Shuntaro; Brazier, John A.; Sugimoto, Naoki

2017-01-01

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases. PMID:28827350

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

PubMed

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.

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

PubMed

Tognetti, Silvia; Riera, Alberto; Speck, Christian

2015-03-01

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

Replication-dependent histone genes are actively transcribed in differentiating and aging retinal neurons

PubMed Central

Banday, Abdul Rouf; Baumgartner, Marybeth; Al Seesi, Sahar; Karunakaran, Devi Krishna Priya; Venkatesh, Aditya; Congdon, Sean; Lemoine, Christopher; Kilcollins, Ashley M; Mandoiu, Ion; Punzo, Claudio; Kanadia, Rahul N

2014-01-01

In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as “replication-dependent.” Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons. PMID:25486194

Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

PubMed Central

Reddy, Kaalak; Schmidt, Monika H.M.; Geist, Jaimie M.; Thakkar, Neha P.; Panigrahi, Gagan B.; Wang, Yuh-Hwa; Pearson, Christopher E.

2014-01-01

R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats. PMID:25147206

R-loop-mediated genomic instability is caused by impairment of replication fork progression

PubMed Central

Gan, Wenjian; Guan, Zhishuang; Liu, Jie; Gui, Ting; Shen, Keng; Manley, James L.; Li, Xialu

2011-01-01

Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans. PMID:21979917

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia

PubMed Central

Cole, John J.; Nelson, David M.; Dikovskaya, Dina; Faller, William J.; Vizioli, Maria Grazia; Hewitt, Rachael N.; Anannya, Orchi; McBryan, Tony; Manoharan, Indrani; van Tuyn, John; Morrice, Nicholas; Pchelintsev, Nikolay A.; Ivanov, Andre; Brock, Claire; Drotar, Mark E.; Nixon, Colin; Clark, William; Sansom, Owen J.; Anderson, Kurt I.; King, Ayala; Blyth, Karen

2014-01-01

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression. PMID:25512559

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia.

PubMed

Rai, Taranjit Singh; Cole, John J; Nelson, David M; Dikovskaya, Dina; Faller, William J; Vizioli, Maria Grazia; Hewitt, Rachael N; Anannya, Orchi; McBryan, Tony; Manoharan, Indrani; van Tuyn, John; Morrice, Nicholas; Pchelintsev, Nikolay A; Ivanov, Andre; Brock, Claire; Drotar, Mark E; Nixon, Colin; Clark, William; Sansom, Owen J; Anderson, Kurt I; King, Ayala; Blyth, Karen; Adams, Peter D

2014-12-15

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression. © 2014 Rai et al.; Published by Cold Spring Harbor Laboratory Press.

FANCJ promotes DNA synthesis through G-quadruplex structures

PubMed Central

Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

2014-01-01

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes. PMID:25193968

The Intolerance of Uncertainty Index: Replication and Extension with an English Sample

ERIC Educational Resources Information Center

Carleton, R. Nicholas; Gosselin, Patrick; Asmundson, Gordon J. G.

2010-01-01

Intolerance of uncertainty (IU) is related to anxiety, depression, worry, and anxiety sensitivity. Precedent IU measures were criticized for psychometric instability and redundancy; alternative measures include the novel 45-item measure (Intolerance of Uncertainty Index; IUI). The IUI was developed in French with 2 parts, assessing general…

Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression.

PubMed

Hartford, Suzanne A; Luo, Yunhai; Southard, Teresa L; Min, Irene M; Lis, John T; Schimenti, John C

2011-10-25

Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.

Specificity of Affective Instability in Patients With Borderline Personality Disorder Compared to Posttraumatic Stress Disorder, Bulimia Nervosa, and Healthy Controls

PubMed Central

Santangelo, Philip; Mussgay, Lutz; Sawitzki, Günther; Trull, Timothy J.; Reinhard, Iris; Steil, Regina; Klein, Christoph; Bohus, Martin; Ebner-Priemer, Ulrich W.

2014-01-01

Affective instability is a core feature of borderline personality disorder (BPD). The use of advanced assessment methodologies and appropriate statistical analyses has led to consistent findings that indicate a heightened instability in patients with BPD compared with healthy controls. However, few studies have investigated the specificity of affective instability among patients with BPD with regard to relevant clinical control groups. In this study, 43 patients with BPD, 28 patients with posttraumatic stress disorder (PTSD), 20 patients with bulimia nervosa (BN), and 28 healthy controls carried e-diaries for 24 hours and were prompted to rate their momentary affective states approximately every 15 minutes while awake. To quantify instability, we used 3 state-of-the-art indices: multilevel models for squared successive differences (SSDs), multilevel models for probability of acute changes (PACs), and aggregated point-by-point changes (APPCs). Patients with BPD displayed heightened affective instability for emotional valence and distress compared with healthy controls, regardless of the specific instability indices. These results directly replicate earlier studies. However, affective instability did not seem to be specific to patients with BPD. With regard to SSDs, PACs, and APPCs, patients with PTSD or BN showed a similar heightened instability of affect (emotional valence and distress) to that of patients with BPD. Our results give raise to the discussion if affective instability is a transdiagnostic or a disorder-specific mechanism. Current evidence cannot answer this question, but investigating psychopathological mechanisms in everyday life across disorders is a promising approach to enhance validity and specificity of mental health diagnoses. PMID:24661176

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

NASA Astrophysics Data System (ADS)

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.

Recovery from the DNA Replication Checkpoint

PubMed Central

Chaudhury, Indrajit; Koepp, Deanna M.

2016-01-01

Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838

Parametric dependence of density limits in the Tokamak Experiment for Technology Oriented Research (TEXTOR): Comparison of thermal instability theory with experiment

NASA Astrophysics Data System (ADS)

Kelly, F. A.; Stacey, W. M.; Rapp, J.

2001-11-01

The observed dependence of the TEXTOR [Tokamak Experiment for Technology Oriented Research: E. Hintz, P. Bogen, H. A. Claassen et al., Contributions to High Temperature Plasma Physics, edited by K. H. Spatschek and J. Uhlenbusch (Akademie Verlag, Berlin, 1994), p. 373] density limit on global parameters (I, B, P, etc.) and wall conditioning is compared with the predicted density limit parametric scaling of thermal instability theory. It is necessary first to relate the edge parameters of the thermal instability theory to n¯ and the other global parameters. The observed parametric dependence of the density limit in TEXTOR is generally consistent with the predicted density limit scaling of thermal instability theory. The observed wall conditioning dependence of the density limit can be reconciled with the theory in terms of the radiative emissivity temperature dependence of different impurities in the plasma edge. The thermal instability theory also provides an explanation of why symmetric detachment precedes radiative collapse for most low power shots, while a multifaceted asymmetric radiation from the edge MARFE precedes detachment for most high power shots.

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

DOE PAGES

Garbe, James C.; Vrba, Lukas; Sputova, Klara; ...

2014-10-29

Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16INK4; replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agentsmore » are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional “passenger” errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.« less

Fanconi anaemia and the repair of Watson and Crick DNA crosslinks.

PubMed

Kottemann, Molly C; Smogorzewska, Agata

2013-01-17

The function of Fanconi anaemia proteins is to maintain genomic stability. Their main role is in the repair of DNA interstrand crosslinks, which, by covalently binding the Watson and the Crick strands of DNA, impede replication and transcription. Inappropriate repair of interstrand crosslinks causes genomic instability, leading to cancer; conversely, the toxicity of crosslinking agents makes them a powerful chemotherapeutic. Fanconi anaemia proteins can promote stem-cell function, prevent tumorigenesis, stabilize replication forks and inhibit inaccurate repair. Recent advances have identified endogenous aldehydes as possible culprits of DNA damage that may induce the phenotypes seen in patients with Fanconi anaemia.

Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

DTIC Science & Technology

2012-10-11

mismatched base stacks with a conserved phenylalanine in Msh6, and/or (iii) DNA flexibility, since MutSa-bound mismatched DNA is kinked, and a...AB, Watt DL , Watts BE, et al. (2010) Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol 6: 774–781. 24. Poloumienko A

Understanding Chinese American Adolescents' Developmental Outcomes: Insights from the Family Stress Model

ERIC Educational Resources Information Center

Benner, Aprile D.; Kim, Su Yeong

2010-01-01

In this brief report, we investigated whether the Family Stress Model could be replicated with a sample of Chinese American families. Path analyses with 444 adolescents and their parents provided support for the model's generalizability. Specifically, mothers' and fathers' reports of economic status (i.e., income, financial, and job instability)…

Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability

DOE PAGES

Tsutakawa, Susan E.; Thompson, Mark J.; Arvai, Andrew S.; ...

2017-06-27

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA formore » catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.« less

Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability

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

Tsutakawa, Susan E.; Thompson, Mark J.; Arvai, Andrew S.

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA formore » catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.« less

Telomere length maintenance--an ALTernative mechanism.

PubMed

Royle, N J; Foxon, J; Jeyapalan, J N; Mendez-Bermudez, A; Novo, C L; Williams, J; Cotton, V E

2008-01-01

The Alternative Lengthening of Telomeres (ALT) mechanism is utilised by approximately 10% of human tumours and a higher proportion of some types of sarcomas. ALT+ cell lines and tumours show heterogeneous telomere length, extra-chromosomal circular and linear telomeric DNA, ALT associated promyelocytic bodies (APBs), a high frequency of post-replication exchanges in telomeres (designated as telomere-sister chromatid exchanges, T-SCE) and high instability at a GC-rich minisatellite, MS32 (D1S8). It is clear that there is a link between the minisatellite instability and the mechanism that underpins ALT, however currently the nature of this relationship is uncertain. Single molecule analysis of telomeric DNA from ALT+ cell lines and tumours has revealed complex telomere mutations that have not been seen in cell lines or tumours that express telomerase. These complex telomere mutations cannot be explained by T-SCE but must arise by another inter-molecular process. The break-induced replication (BIR) model that may explain the observed high frequency of T-SCE and the presence of complex telomere mutations is reviewed. Copyright 2008 S. Karger AG, Basel.

Claims-based proxies of patient instability among commercially insured adults with schizophrenia

PubMed Central

Ruetsch, Charles; Un, Hyong; Waters, Heidi C

2018-01-01

Objective Schizophrenia (Sz) patients are among the highest utilizers of hospital-based services. Prevention of relapse is in part a treatment goal in order to reduce hospital admissions. However, predicting relapse is a challenge, particularly for payers and disease management firms with only access to claims data. Understandably, such organizations have had little success predicting relapse. A tool that allows payers to identify patients at elevated risk of relapse could facilitate targeted interventions prior to relapse and avoid rehospitalization. In this study, a series of proxy measures of patient instability, calculated from claims data were examined for their utility in identifying Sz patients at elevated risk of relapse. Methods Aetna claims were used to assess the relationship between instability of Sz patients and valence and magnitude of antipsychotic (AP) medication change during a 2-year period. Six proxies of instability including hospital admissions, emergency department visits, medication utilization patterns, and use of outpatient services were identified. Results were replicated using claims data from Truven MarketScan®. Results Patients who switched AP ingredient had the highest overall instability at the point of switch and the second steepest decline in instability following switch. Those who changed to a long-acting injectable AP showed the second highest level of instability and the steepest decrease in instability following the change. Patients augmented with a second AP showed the smallest increase in instability, up to the switch. Results were directionally consistent between the two data sets. Conclusion Using claims-based proxy measures to estimate instability may provide a viable method to better understand Sz patient markers of change in disease severity. Also, such proxies could be used to identify those individuals with the greatest need for treatment modification preventing relapse, improving patient outcomes, and reducing the burden of illness. PMID:29765242

Saving the Inner Solar System with an Early Instability

NASA Astrophysics Data System (ADS)

Clement, Matthew; Kaib, Nathan A.; Raymond, Sean N.; Walsh, Kevin J.

2018-04-01

An orbital instability between the solar system’s giant planets (the so-called Nice Model) has been shown to greatly disturb the orbits of the young terrestrial planets. Undesirable outcomes such as over-excitated orbits, ejections and collisions can be avoided if the instability occurs before the inner planets are fully formed. Such a scenario also has the advantage of limiting the mass and formation time of Mars when it occurs within several million years (Myr) of gas disk dissipation. The dynamical effects of the instability cause many small embryos and planetesimals to scatter away from the forming Mars, and lead to heavy mass depletion in the Asteroid Belt. We present new simulations of this scenario that demonstrate its ability to accurately reproduce the eccentricity, inclination and resonant structures of the Asteroid Belt. Furthermore, we perform simulations using an integration scheme which accounts for the fragmentation of colliding bodies. The final terrestrial systems formed in these simulations provide a better match to the actual planets' compact mass distribution and dynamically cold orbits. An early instability scenario is thus very successful at simultaneously replicating the dynamical state of both the inner and outer solar system.

Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

PubMed Central

On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

2014-01-01

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

The annealing helicase and branch migration activities of Drosophila HARP.

PubMed

Kassavetis, George A; Kadonaga, James T

2014-01-01

HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. We found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.

Applications of Replicating-Competent Reporter-Expressing Viruses in Diagnostic and Molecular Virology.

PubMed

Li, Yongfeng; Li, Lian-Feng; Yu, Shaoxiong; Wang, Xiao; Zhang, Lingkai; Yu, Jiahui; Xie, Libao; Li, Weike; Ali, Razim; Qiu, Hua-Ji

2016-05-06

Commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for antivirals, as well as for tracking viral proteins or virus transport in real time. Notably, the development of replicating-competent reporter-expressing viruses (RCREVs) has provided an excellent option to detect directly viral replication without the use of secondary labeling, which represents a significant advance in virology. This article reviews the applications of RCREVs in diagnostic and molecular virology, including rapid neutralization tests, high-throughput screening systems, identification of viral receptors and virus-host interactions, dynamics of viral infections in vitro and in vivo, vaccination approaches and others. However, there remain various challenges associated with RCREVs, including pathogenicity alterations due to the insertion of a reporter gene, instability or loss of the reporter gene expression, or attenuation of reporter signals in vivo. Despite all these limitations, RCREVs have become powerful tools for both basic and applied virology with the development of new technologies for generating RCREVs, the inventions of novel reporters and the better understanding of regulation of viral replication.

Genome health nutrigenomics and nutrigenetics--diagnosis and nutritional treatment of genome damage on an individual basis.

PubMed

Fenech, Michael

2008-04-01

The term nutrigenomics refers to the effect of diet on gene expression. The term nutrigenetics refers to the impact of inherited traits on the response to a specific dietary pattern, functional food or supplement on a specific health outcome. The specific fields of genome health nutrigenomics and genome health nutrigenetics are emerging as important new research areas because it is becoming increasingly evident that (a) risk for developmental and degenerative disease increases with DNA damage which in turn is dependent on nutritional status and (b) optimal concentration of micronutrients for prevention of genome damage is also dependent on genetic polymorphisms that alter function of genes involved directly or indirectly in uptake and metabolism of micronutrients required for DNA repair and DNA replication. Development of dietary patterns, functional foods and supplements that are designed to improve genome health maintenance in humans with specific genetic backgrounds may provide an important contribution to a new optimum health strategy based on the diagnosis and individualised nutritional treatment of genome instability i.e. Genome Health Clinics.

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

PubMed

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

Epstein-Barr Virus Hijacks DNA Damage Response Transducers to Orchestrate Its Life Cycle.

PubMed

Hau, Pok Man; Tsao, Sai Wah

2017-11-16

The Epstein-Barr virus (EBV) is a ubiquitous virus that infects most of the human population. EBV infection is associated with multiple human cancers, including Burkitt's lymphoma, Hodgkin's lymphoma, a subset of gastric carcinomas, and almost all undifferentiated non-keratinizing nasopharyngeal carcinoma. Intensive research has shown that EBV triggers a DNA damage response (DDR) during primary infection and lytic reactivation. The EBV-encoded viral proteins have been implicated in deregulating the DDR signaling pathways. The consequences of DDR inactivation lead to genomic instability and promote cellular transformation. This review summarizes the current understanding of the relationship between EBV infection and the DDR transducers, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase), and discusses how EBV manipulates the DDR signaling pathways to complete the replication process of viral DNA during lytic reactivation.

Emergence of multicellular organisms with dynamic differentiation and spatial pattern.

PubMed

Furusawa, C; Kaneko, K

1998-01-01

The origin of multicellular organisms and the mechanism of development in cell societies are studied by choosing a model with intracellular biochemical dynamics allowing for oscillations, cell-cell interaction through diffusive chemicals on a two-dimensional grid, and state-dependent cell adhesion. Cells differentiate due to a dynamical instability, as described by our "isologous diversification" theory. A fixed spatial pattern of differentiated cells emerges, where spatial information is sustained by cell-cell interactions. This pattern is robust against perturbations. With an adequate cell adhesion force, active cells are release that form the seed of a new generation of multicellular organisms, accompanied by death of the original multicellular unit as a halting state. It is shown that the emergence of multicellular organisms with differentiation, regulation, and life cycle is not an accidental event, but a natural consequence in a system of replicating cells with growth.

Defining the steps that lead to cancer: replicative telomere erosion, aneuploidy and an epigenetic maturation arrest of tissue stem cells.

PubMed

Stindl, Reinhard

2008-01-01

Recently, an influential sequencing study found that more than 1700 genes had non-silent mutations in either a breast or colorectal cancer, out of just 11 breast and 11 colorectal tumor samples. This is not surprising given the fact that genomic instability is the hallmark of cancer cells. The plethora of genomic alterations found in every carcinoma does not obey the 'law of genotype-phenotype correlation', since the same histological subtype of cancer harbors different gene mutations and chromosomal aberrations in every patient. In an attempt to make sense out of the observed genetic and chromosomal chaos in cancer, I propose a cascade model. According to this model, tissue regeneration depends on the proliferation and serial activation of stem cells. Replicative telomere erosion limits the proliferative life span of adult stem cells and results in the Hayflick limit (M1). However, local tissue exhaustion or old age might promote the activation of M1-deficient tissue stem cells. Extended proliferation of these cells leads to telomere-driven chromosomal instability and aneuploidy (abnormal balance of chromosomes and/or chromosome material). Several of the aforementioned steps have been already described in the literature. However, in contrast to common theories, it is proposed here that the genomic damage blocks the epigenetic differentiation switch. As a result of aneuploidy, differentiation-specific genes cannot be activated by modification of methylation patterns. Consequently, the phenotype of cancer tissue is largely determined by the epigenetic maturation arrest of tissue stem cells, which in addition enables a fraction of cancer cells to proliferate, invade and metastasize, as normal adult stem cells do. The new model combines genetic and epigenetic alterations of cancer cells in one causative cascade and offers an explanation for why identical histologic cancer types harbor a confusing variety of chromosomal and gene aberrations. The Viennese Cascade, as presented here, may end the debate on if and how 'tumor-unspecific' aneuploidy leads to cancer.

Molecular Signature and Mechanisms of Hepatitis D Virus-Associated Hepatocellular Carcinoma.

PubMed

Diaz, Giacomo; Engle, Ronald E; Tice, Ashley; Melis, Marta; Montenegro, Stephanie; Rodriguez-Canales, Jaime; Hanson, Jeffrey; Emmert-Buck, Michael R; Bock, Kevin W; Moore, Ian N; Zamboni, Fausto; Govindarajan, Sugantha; Kleiner, David; Farci, Patrizia

2018-06-01

There is limited data on the molecular mechanisms whereby hepatitis D virus (HDV) promotes liver cancer. Therefore, serum and liver specimens obtained at the time of liver transplantation from well-characterized patients with HDV-HCC (n-5) and with non-HCC HDV cirrhosis (n=7) were studied using an integrated genomic approach. Transcriptomic profiling was performed using laser capture-microdissected (LCM) malignant and non-malignant hepatocytes, tumorous and non-tumorous liver tissue from patients with HDV-HCC, and liver tissue from patients with non-HCC HDV cirrhosis. HDV-HCC was also compared with hepatitis B virus (HBV) HBV-HCC alone and hepatitis C virus (HCV) HCV-HCC. HDV malignant hepatocytes were characterized by an enrichment of up-regulated transcripts associated with pathways involved in cell cycle/DNA replication, damage and repair (sonic hedgehog, GADD45, DNA-damage-induced 14-3-3σ, cyclins and cell cycle regulation, cell cycle: G2/M DNA-damage checkpoint regulation, and hereditary breast cancer). Moreover, a large network of genes identified functionally relate to DNA repair, cell cycle, mitotic apparatus and cell division, including 4 cancer testis antigen genes, attesting to the critical role of genetic instability in this tumor. Besides being over-expressed, these genes were also strongly co-regulated. Gene co-regulation was high not only when compared to non-malignant hepatocytes, but also to malignant hepatocytes from HBV-HCC alone or HCV-HCC. Activation and co-regulation of genes critically associated with DNA replication, damage, and repair point to genetic instability as an important mechanism of HDV hepatocarcinogenesis. This specific HDV-HCC trait emerged also from the comparison of the molecular pathways identified for each hepatitis virus-associated HCC. Despite the dependence of HDV on HBV, these findings suggest that HDV and HBV promote carcinogenesis by distinct molecular mechanisms. This study identifies a molecular signature of HDV-associated hepatocellular carcinoma and suggests the potential for new biomarkers for early diagnostics. Copyright ©2018, American Association for Cancer Research.

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

PubMed Central

Hamperl, Stephan; Cimprich, Karlene A.

2014-01-01

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

Therapeutic targeting of replicative immortality

PubMed Central

Yaswen, Paul; MacKenzie, Karen L.; Keith, W. Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L.; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S.; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I.; Azmi, Asfar S.; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S. Salman; Nowsheen, Somaira; Yang, Xujuan

2015-01-01

One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed “senescence,” can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells’ heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. PMID:25869441

YAP controls retinal stem cell DNA replication timing and genomic stability

PubMed Central

Cabochette, Pauline; Vega-Lopez, Guillermo; Bitard, Juliette; Parain, Karine; Chemouny, Romain; Masson, Christel; Borday, Caroline; Hedderich, Marie; Henningfeld, Kristine A; Locker, Morgane; Bronchain, Odile; Perron, Muriel

2015-01-01

The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability. DOI: http://dx.doi.org/10.7554/eLife.08488.001 PMID:26393999

Viscous Analysis of Pulsating Hydrodynamic Instability and Thermal Coupling Liquid-Propellant Combustion

NASA Technical Reports Server (NTRS)

Margolis, Stephen B.; Sacksteder, Kurt (Technical Monitor)

2000-01-01

A pulsating form of hydrodynamic instability has recently been shown to arise during liquid-propellant deflagration in those parameter regimes where the pressure-dependent burning rate is characterized by a negative pressure sensitivity. This type of instability can coexist with the classical cellular, or Landau form of hydrodynamic instability, with the occurrence of either dependent on whether the pressure sensitivity is sufficiently large or small in magnitude. For the inviscid problem, it has been shown that, when the burning rate is realistically allowed to depend on temperature as well as pressure, sufficiently large values of the temperature sensitivity relative to the pressure sensitivity causes like pulsating form of hydrodynamic instability to become dominant. In that regime, steady, planar burning becomes intrinsically unstable to pulsating disturbances whose wave numbers are sufficiently small. This analysis is extended to the fully viscous case, where it is shown that although viscosity is stabilizing for intermediate and larger wave number perturbations, the intrinsic pulsating instability for small wave numbers remains. Under these conditions, liquid-propellant combustion is predicted to be characterized by large unsteady cells along the liquid/gas interface.

Polarization Signatures of Kink Instabilities in the Blazar Emission Region from Relativistic Magnetohydrodynamic Simulations

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

Zhang, Haocheng; Taylor, Greg; Li, Hui

Kink instabilities are likely to occur in the current-carrying magnetized plasma jets. Recent observations of the blazar radiation and polarization signatures suggest that the blazar emission region may be considerably magnetized. While the kink instability has been studied with first-principle magnetohydrodynamic (MHD) simulations, the corresponding time-dependent radiation and polarization signatures have not been investigated. In this paper, we perform comprehensive polarization-dependent radiation modeling of the kink instability in the blazar emission region based on relativistic MHD (RMHD) simulations. We find that the kink instability may give rise to strong flares with polarization angle (PA) swings or weak flares with polarizationmore » fluctuations, depending on the initial magnetic topology and magnetization. These findings are consistent with observations. Compared with the shock model, the kink model generates polarization signatures that are in better agreement with the general polarization observations. Therefore, we suggest that kink instabilities may widely exist in the jet environment and provide an efficient way to convert the magnetic energy and produce multiwavelength flares and polarization variations.« less

Polarization Signatures of Kink Instabilities in the Blazar Emission Region from Relativistic Magnetohydrodynamic Simulations

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

Zhang, Haocheng; Li, Hui; Guo, Fan

Kink instabilities are likely to occur in the current-carrying magnetized plasma jets. Recent observations of the blazar radiation and polarization signatures suggest that the blazar emission region may be considerably magnetized. While the kink instability has been studied with first-principle magnetohydrodynamic (MHD) simulations, the corresponding time-dependent radiation and polarization signatures have not been investigated. Here, in this paper, we perform comprehensive polarization-dependent radiation modeling of the kink instability in the blazar emission region based on relativistic MHD (RMHD) simulations. We find that the kink instability may give rise to strong flares with polarization angle (PA) swings or weak flares withmore » polarization fluctuations, depending on the initial magnetic topology and magnetization. These findings are consistent with observations. In addition, compared with the shock model, the kink model generates polarization signatures that are in better agreement with the general polarization observations. Therefore, we suggest that kink instabilities may widely exist in the jet environment and provide an efficient way to convert the magnetic energy and produce multiwavelength flares and polarization variations.« less

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

NASA Technical Reports Server (NTRS)

Schwartz, Jeffrey L.; Jordan, Robert; Evans, Helen H.; Lenarczyk, Marek; Liber, Howard

2003-01-01

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

PubMed Central

Lathrop, Stephanie K.; Binder, Kelsey A.; Starr, Tregei; Cooper, Kendal G.; Chong, Audrey; Carmody, Aaron B.

2015-01-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. PMID:25895967

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

PubMed

Lathrop, Stephanie K; Binder, Kelsey A; Starr, Tregei; Cooper, Kendal G; Chong, Audrey; Carmody, Aaron B; Steele-Mortimer, Olivia

2015-07-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

PubMed

Hilton, Benjamin A; Liu, Ji; Cartwright, Brian M; Liu, Yiyong; Breitman, Maya; Wang, Youjie; Jones, Rowdy; Tang, Hui; Rusinol, Antonio; Musich, Phillip R; Zou, Yue

2017-09-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin). We previously reported that XPA mislocalized to the progerin-induced DNA double-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation, DNA damage responses, and early replication arrest in HGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNA at replication forks. Our results suggest that although PCNA is much more competitive than XPA in binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPA-replication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in HGPS.-Hilton, B. A., Liu, J., Cartwright, B. M., Liu, Y., Breitman, M., Wang, Y., Jones, R., Tang, H., Rusinol, A., Musich, P. R., Zou, Y. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes. © FASEB.

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

PubMed

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, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.

A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

PubMed

Spoerri, Loredana; Brooks, Kelly; Chia, KeeMing; Grossman, Gavriel; Ellis, Jonathan J; Dahmer-Heath, Mareike; Škalamera, Dubravka; Pavey, Sandra; Burmeister, Bryan; Gabrielli, Brian

2016-05-01

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed

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.

On the interrelation of divergence, flutter and auto-parametric resonance.

NASA Technical Reports Server (NTRS)

Herrmann, G.; Hauger, W.

1973-01-01

The dependence between static instability and kinetic instability (flutter) on autoparameteric resonance is studied by taking compressibility into account in a model of a cantilever beam under the action of a follower force. It is shown that both instabilities are formally special cases of instabilities known as subharmonic and combination resonances.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

PubMed Central

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

Shoulder Instability

MedlinePlus

... as bad as the pain of a sudden injury. Your shoulder might be sore when you move it. It ... Treatment How is shoulder instability treated? Treatment for shoulder instability depends on how bad your injury is and how important it is for you ...

Instability timescale for the inclination instability in the solar system

NASA Astrophysics Data System (ADS)

Zderic, Alexander; Madigan, Ann-Marie; Fleisig, Jacob

2018-04-01

The gravitational influence of small bodies is often neglected in the study of solar system dynamics. However, this is not always an appropriate assumption. For example, mutual secular torques between low mass particles on eccentric orbits can result in a self-gravity instability (`inclination instability'; Madigan & McCourt 2016). During the instability, inclinations increase exponentially, eccentricities decrease (detachment), and orbits cluster in argument of perihelion. In the solar system, the orbits of the most distant objects show all three of these characteristics (high inclination: Volk & Malhotra (2017), detachment: Delsanti & Jewitt (2006), and argument of perihelion clustering: Trujillo & Sheppard (2014)). The inclination instability is a natural explanation for these phenomena.Unfortunately, full N-body simulations of the solar system are unfeasible (N ≈ O(1012)), and the behavior of the instability depends on N, prohibiting the direct application of lower N simulations. Here we present the instability timescale's functional dependence on N, allowing us to extrapolate our simulation results to that appropriate for the solar system. We show that ~5 MEarth of small icy bodies in the Sedna region is sufficient for the inclination instability to occur in the outer solar system.

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

PubMed

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.

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

PubMed

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.

Gravitational Waves from Neutron Stars

NASA Astrophysics Data System (ADS)

Kokkotas, Konstantinos

2016-03-01

Neutron stars are the densest objects in the present Universe, attaining physical conditions of matter that cannot be replicated on Earth. These unique and irreproducible laboratories allow us to study physics in some of its most extreme regimes. More importantly, however, neutron stars allow us to formulate a number of fundamental questions that explore, in an intricate manner, the boundaries of our understanding of physics and of the Universe. The multifaceted nature of neutron stars involves a delicate interplay among astrophysics, gravitational physics, and nuclear physics. The research in the physics and astrophysics of neutron stars is expected to flourish and thrive in the next decade. The imminent direct detection of gravitational waves will turn gravitational physics into an observational science, and will provide us with a unique opportunity to make major breakthroughs in gravitational physics, in particle and high-energy astrophysics. These waves, which represent a basic prediction of Einstein's theory of general relativity but have yet to be detected directly, are produced in copious amounts, for instance, by tight binary neutron star and black hole systems, supernovae explosions, non-axisymmetric or unstable spinning neutron stars. The focus of the talk will be on the neutron star instabilities induced by rotation and the magnetic field. The conditions for the onset of these instabilities and their efficiency in gravitational waves will be presented. Finally, the dependence of the results and their impact on astrophysics and especially nuclear physics will be discussed.

SCF(FBXW7α) modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability.

PubMed

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2014-06-30

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.

SCFFBXW7α modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability

PubMed Central

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

2014-01-01

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint. PMID:24970797

Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

PubMed Central

Yeeles, Joseph T.P.; Deegan, Tom D.; Janska, Agnieszka; Early, Anne; Diffley, John F. X.

2016-01-01

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

Regulated eukaryotic DNA replication origin firing with purified proteins.

PubMed

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Viscous and Thermal Effects on Hydrodynamic Instability in Liquid-Propellant Combustion

NASA Technical Reports Server (NTRS)

Margolis, Stephen B.; Sacksteder, Kurt (Technical Monitor)

2000-01-01

A pulsating form of hydrodynamic instability has recently been shown to arise during the deflagration of liquid propellants in those parameter regimes where the pressure-dependent burning rate is characterized by a negative pressure sensitivity. This type of instability can coexist with the classical cellular, or Landau, form of hydrodynamic instability, with the occurrence of either dependent on whether the pressure sensitivity is sufficiently large or small in magnitude. For the inviscid problem, it has been shown that when the burning rate is realistically allowed to depend on temperature as well as pressure, that sufficiently large values of the temperature sensitivity relative to the pressure sensitivity causes the pulsating form of hydrodynamic instability to become dominant. In that regime, steady, planar burning becomes intrinsically unstable to pulsating disturbances whose wavenumbers are sufficiently small. In the present work, this analysis is extended to the fully viscous case, where it is shown that although viscosity is stabilizing for intermediate and larger wavenumber perturbations, the intrinsic pulsating instability for small wavenumbers remains. Under these conditions, liquid-propellant combustion is predicted to be characterized by large unsteady cells along the liquid/gas interface.

Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

PubMed

Morafraile, Esther C; Diffley, John F X; Tercero, José Antonio; Segurado, Mónica

2015-01-20

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.

Bloom’s Syndrome: Why Not Premature Aging? A comparison of the BLM and WRN helicases

PubMed Central

de Renty, Christelle; Ellis, Nathan A.

2016-01-01

Genomic instability is a hallmark of cancer and aging. Premature aging (progeroid) syndromes are often caused by mutations in genes whose function is to ensure genomic integrity. The RecQ family of DNA helicases is highly conserved and plays crucial roles as genome caretakers. In human, mutations in three RecQ genes — BLM, WRN, and RECQL4 — give rise to Bloom’s syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson’s syndrome (RTS), respectively. WS is a prototypic premature aging disorder; however, the clinical features present in BS and RTS do not indicate accelerated aging. The BLM helicase has pivotal functions at the crossroads of DNA replication, recombination, and repair. BS cells exhibit a characteristic form of genomic instability that includes excessive recombination. The excessive homologous recombination drives the development of the many types of cancers that affect persons in the normal population. Replication delay and slower cell turnover rates have been proposed to explain many features of Bloom’s syndrome, such as short stature. More recently, aberrant transcriptional regulation of growth and survival genes has been proposed as a hypothesis. PMID:27238185

The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

PubMed Central

Patel, Prasanta K.; Kommajosyula, Naveen; Rosebrock, Adam; Bensimon, Aaron; Leatherwood, Janet; Bechhoefer, John

2008-01-01

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency. PMID:18799612

RTEL1 contributes to DNA replication and repair and telomere maintenance.

PubMed

Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M

2012-07-01

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.

A 2-DOF microstructure-dependent model for the coupled torsion/bending instability of rotational nanoscanner

NASA Astrophysics Data System (ADS)

Keivani, M.; Abadian, N.; Koochi, A.; Mokhtari, J.; Abadyan, M.

2016-10-01

It has been well established that the physical performance of nanodevices might be affected by the microstructure. Herein, a two-degree-of-freedom model base on the modified couple stress theory is developed to incorporate the impact of microstructure in the torsion/bending coupled instability of rotational nanoscanner. Effect of microstructure dependency on the instability parameters is determined as a function of the microstructure parameter, bending/torsion coupling ratio, van der Waals force parameter and geometrical dimensions. It is found that the bending/torsion coupling substantially affects the stable behavior of the scanners especially those with long rotational beam elements. Impact of microstructure on instability voltage of the nanoscanner depends on coupling ratio and the conquering bending mode over torsion mode. This effect is more highlighted for higher values of coupling ratio. Depending on the geometry and material characteristics, the presented model is able to simulate both hardening behavior (due to microstructure) and softening behavior (due to torsion/bending coupling) of the nanoscanners.

Further evidences for sleep instability and impaired spindle-delta dynamics in schizophrenia: a whole-night polysomnography study with neuroloop-gain and sleep-cycle analysis.

PubMed

Sasidharan, Arun; Kumar, Sunil; Nair, Ajay Kumar; Lukose, Ammu; Marigowda, Vrinda; John, John P; Kutty, Bindu M

2017-10-01

Sleep offers a unique window into the brain dysfunctions in schizophrenia. Many past sleep studies have reported abnormalities in both macro-sleep architecture (like increased awakenings) as well as micro-sleep-architecture (like spindle deficits) in patients with schizophrenia (PSZ). The present study attempts to replicate previous reports of macro- and micro-sleep-architectural abnormalities in schizophrenia. In addition, the study also examined sleep-stage changes and spindle-delta dynamics across sleep-cycles to provide further evidence in support of the dysfunctional thalamocortical mechanisms causing sleep instability and poor sleep maintenance associated with schizophrenia pathophysiology. Whole-night polysomnography was carried out among 45 PSZ and 39 age- and gender-matched healthy control subjects. Sleep-stage dynamics were assessed across sleep-cycles using a customized software algorithm. Spindle-delta dynamics across sleep-cycles were determined using neuroloop-gain analysis. PSZ showed macro-sleep architecture abnormalities such as prolonged sleeplessness, increased intermittent-awakenings, long sleep-onset latency, reduced non-rapid eye movement (NREM) stage 2 sleep, increased stage transitions, and poor sleep efficiency. They also showed reduced spindle density (sigma neuroloop-gain) but comparable slow wave density (delta neuroloop-gain) throughout the sleep. Sleep-cycle-wise analysis revealed transient features of sleep instability due to significantly increased intermittent awakenings especially in the first and third sleep-cycles, and unstable and recurrent stage transitions in both NREM (first sleep-cycle) and rapid eye movement (REM) sleep-periods (second sleep-cycle). Spindle deficits were persistent across the first three cycles and were positively correlated with sleep disruption during the subsequent REM sleep. In addition to replicating previously reported sleep deficits in PSZ, the current study showed subtle deficits in NREM-REM alterations across whole-night polysomnography. These results point towards a possible maladaptive interplay between unstable thalamocortical networks, resulting in sleep-cycle-specific instability patterns associated with schizophrenia pathophysiology. Copyright © 2017 Elsevier B.V. All rights reserved.

The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

PubMed

O'Driscoll, Mark

2017-01-01

Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Critical Layers and Protoplanetary Disk Turbulence

NASA Astrophysics Data System (ADS)

Umurhan, Orkan M.; Shariff, Karim; Cuzzi, Jeffrey N.

2016-10-01

A linear analysis of the zombie vortex instability (ZVI) is performed in a stratified shearing sheet setting for three model barotropic shear flows. The linear analysis is done by utilizing a Green’s function formulation to resolve the critical layers of the associated normal-mode problem. The instability is the result of a resonant interaction between a Rossby wave and a gravity wave that we refer to as Z-modes. The associated critical layer is the location where the Doppler-shifted frequency of a distant Rossby wave equals the local Brunt-Väisälä frequency. The minimum required Rossby number for instability, {\\mathtt{Ro}}=0.2, is confirmed for parameter values reported in the literature. It is also found that the shear layer supports the instability in the limit where stratification vanishes. The ZVI is examined in a jet model, finding that the instability can occur for {\\mathtt{Ro}}=0.05. Nonlinear vorticity forcing due to unstable Z-modes is shown to result in the creation of a jet flow at the critical layer emerging as the result of the competition between the vertical lifting of perturbation radial vorticity and the radial transport of perturbation vertical vorticity. We find that the picture of this instability leading to a form of nonlinearly driven self-replicating pattern of creation and destruction is warranted: a parent jet spawns a growing child jet at associated critical layers. A mature child jet creates a next generation of child jets at associated critical layers of the former while simultaneously contributing to its own destruction via the Rossby wave instability.

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

PubMed

Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

2009-08-01

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Particle acceleration magnetic field generation, and emission in Relativistic pair jets

NASA Technical Reports Server (NTRS)

Nishikawa, K.-I.; Ramirez-Ruiz, E.; Hardee, P.; Hededal, C.; Kouveliotou, C.; Fishman, G. J.

2005-01-01

Plasma waves and their associated instabilities (e.g., the Buneman instability, two-streaming instability, and the Weibel instability) are responsible for particle acceleration in relativistic pair jets. Using a 3-D relativistic electromagnetic particle (REMP) code, we have investigated particle acceleration associated with a relativistic pair jet propagating through a pair plasma. Simulations show that the Weibel instability created in the collisionless shock accelerates particles perpendicular and parallel to the jet propagation direction. Simulation results show that this instability generates and amplifies highly nonuniform, small-scale magnetic fields, which contribute to the electron's transverse deflection behind the jet head. The "jitter' I radiation from deflected electrons can have different properties than synchrotron radiation which is calculated in a uniform magnetic field. This jitter radiation may be important to understanding the complex time evolution and/or spectral structure in gamma-ray bursts, relativistic jets, and supernova remnants. The growth rate of the Weibel instability and the resulting particle acceleration depend on the magnetic field strength and orientation, and on the initial particle distribution function. In this presentation we explore some of the dependencies of the Weibel instability and resulting particle acceleration on the magnetic field strength and orientation, and the particle distribution function.

The Time-Dependent Structure of the Electron Reconnection Layer

NASA Technical Reports Server (NTRS)

Hesse, Michael; Zenitani, Seiji; Kuznetsova, Masha; Klimas, Alex

2009-01-01

Collisionless magnetic reconnection is often associated with time-dependent behavior. Specifically, current layers in the diffusion region can become unstable to tearing-type instabilities on one hand, or to instabilities with current-aligned wave vectors on the other. In the former case, the growth of tearing instabilities typically leads to the production of magnetic islands, which potentially provide feedback on the reconnection process itself, as well as on the rate of reconnection. The second class of instabilities tend to modulate the current layer along the direction of the current flow, for instance generating kink-type perturbations, or smaller-scale turbulence with the potential to broaden the current layer. All of these processes contribute to rendering magnetic reconnection time-dependent. In this presentation, we will provide a summary of these effects, and a discussion of how much they contribute to the overall magnetic reconnection rate.

Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy

PubMed Central

Kautz, Tiffany F; Guerbois, Mathilde; Khanipov, Kamil; Yun, Ruimei; Warmbrod, Kelsey L; Fofanov, Yuriy; Weaver, Scott C; Forrester, Naomi L

2018-01-01

Abstract During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy. PMID:29593882

ELF-MF exposure affects the robustness of epigenetic programming during granulopoiesis

NASA Astrophysics Data System (ADS)

Manser, Melissa; Sater, Mohamad R. Abdul; Schmid, Christoph D.; Noreen, Faiza; Murbach, Manuel; Kuster, Niels; Schuermann, David; Schär, Primo

2017-03-01

Extremely-low-frequency magnetic fields (ELF-MF) have been classified as “possibly carcinogenic” to humans on the grounds of an epidemiological association of ELF-MF exposure with an increased risk of childhood leukaemia. Yet, underlying mechanisms have remained obscure. Genome instability seems an unlikely reason as the energy transmitted by ELF-MF is too low to damage DNA and induce cancer-promoting mutations. ELF-MF, however, may perturb the epigenetic code of genomes, which is well-known to be sensitive to environmental conditions and generally deranged in cancers, including leukaemia. We examined the potential of ELF-MF to influence key epigenetic modifications in leukaemic Jurkat cells and in human CD34+ haematopoietic stem cells undergoing in vitro differentiation into the neutrophilic lineage. During granulopoiesis, sensitive genome-wide profiling of multiple replicate experiments did not reveal any statistically significant, ELF-MF-dependent alterations in the patterns of active (H3K4me2) and repressive (H3K27me3) histone marks nor in DNA methylation. However, ELF-MF exposure showed consistent effects on the reproducibility of these histone and DNA modification profiles (replicate variability), which appear to be of a stochastic nature but show preferences for the genomic context. The data indicate that ELF-MF exposure stabilizes active chromatin, particularly during the transition from a repressive to an active state during cell differentiation.

Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

PubMed Central

Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

2017-01-01

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells. PMID:28515305

DNA Damage as a Driver for Growth Delay: Chromosome Instability Syndromes with Intrauterine Growth Retardation

PubMed Central

Hernández-Gómez, Mariana

2017-01-01

DNA is constantly exposed to endogenous and exogenous mutagenic stimuli that are capable of producing diverse lesions. In order to protect the integrity of the genetic material, a wide array of DNA repair systems that can target each specific lesion has evolved. Despite the availability of several repair pathways, a common general program known as the DNA damage response (DDR) is stimulated to promote lesion detection, signaling, and repair in order to maintain genetic integrity. The genes that participate in these pathways are subject to mutation; a loss in their function would result in impaired DNA repair and genomic instability. When the DDR is constitutionally altered, every cell of the organism, starting from development, will show DNA damage and subsequent genomic instability. The cellular response to this is either uncontrolled proliferation and cell cycle deregulation that ensues overgrowth, or apoptosis and senescence that result in tissue hypoplasia. These diverging growth abnormalities can clinically translate as cancer or growth retardation; both features can be found in chromosome instability syndromes (CIS). The analysis of the clinical, cellular, and molecular phenotypes of CIS with intrauterine growth retardation allows inferring that replication alteration is their unifying feature. PMID:29238724

Stabilizing electrochemical interfaces in viscoelastic liquid electrolytes

PubMed Central

2018-01-01

Electrodeposition is a widely practiced method for creating metal, colloidal, and polymer coatings on conductive substrates. In the Newtonian liquid electrolytes typically used, the process is fundamentally unstable. The underlying instabilities have been linked to failure of microcircuits, dendrite formation on battery electrodes, and overlimiting conductance in ion-selective membranes. We report that viscoelastic electrolytes composed of semidilute solutions of very high–molecular weight neutral polymers suppress these instabilities by multiple mechanisms. The voltage window ΔV in which a liquid electrolyte can operate free of electroconvective instabilities is shown to be markedly extended in viscoelastic electrolytes and is a power-law function, ΔV : η1/4, of electrolyte viscosity, η. This power-law relation is replicated in the resistance to ion transport at liquid/solid interfaces. We discuss consequences of our observations and show that viscoelastic electrolytes enable stable electrodeposition of many metals, with the most profound effects observed for reactive metals, such as sodium and lithium. This finding is of contemporary interest for high-energy electrochemical energy storage. PMID:29582017

Cybersickness without the wobble: Experimental results speak against postural instability theory.

PubMed

Dennison, Mark Stephen; D'Zmura, Michael

2017-01-01

It has been suggested that postural instability is necessary for cybersickness to occur. Seated and standing subjects used a head-mounted display to view a virtual tunnel that rotated about their line of sight. We found that the offset direction of perceived vertical settings matched the direction of the tunnel's rotation, so replicating earlier findings. Increasing rotation speed caused cybersickness to increase, but had no significant impact on perceived vertical settings. Postural sway during rotation was similar to postural sway during rest. While a minority of subjects exhibited postural sway in response to the onset of tunnel rotation, the majority did not. Furthermore, cybersickness increased with rotation speed similarly for the seated and standing conditions. Finally, subjects with greater levels of cybersickness exhibited less variation in postural sway. These results lead us to conclude that the link between postural instability and cybersickness is a weak one in the present experiment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Information Cost, Memory Length and Market Instability.

PubMed

Diks, Cees; Li, Xindan; Wu, Chengyao

2018-07-01

In this article, we study the instability of a stock market with a modified version of Diks and Dindo's (2008) model where the market is characterized by nonlinear interactions between informed traders and uninformed traders. In the interaction of heterogeneous agents, we replace the replicator dynamics for the fractions by logistic strategy switching. This modification makes the model more suitable for describing realistic price dynamics, as well as more robust with respect to parameter changes. One goal of our paper is to use this model to explore if the arrival of new information (news) and investor behavior have an effect on market instability. A second, related, goal is to study the way markets absorb new information, especially when the market is unstable and the price is far from being fully informative. We find that the dynamics become locally unstable and prices may deviate far from the fundamental price, routing to chaos through bifurcation, with increasing information costs or decreasing memory length of the uninformed traders.

Overexpressed thioredoxin compensates Fanconi anemia related chromosomal instability.

PubMed

Kontou, Maria; Adelfalk, Caroline; Ramirez, Maria Helena; Ruppitsch, Werner; Hirsch-Kauffmann, Monica; Schweiger, Manfred

2002-04-04

The cause of the molecular defect of Fanconi anemia (FA) remains unknown. Cells from patients with FA exert an elevated spontaneous chromosomal instability which is further triggered by mitomycin C. The induced lability is reduced by overexpression of thioredoxin which is not the case for spontaneous instability. However, both are eliminated by overexpression of thioredoxin cDNA with an added nuclear localization signal. This implies that thioredoxin is lacking in the nuclei of FA cells. The total thioredoxin content in all FA cells tested is reduced. The resultant lack of nuclear thioredoxin can be the explanation for the major symptomatology in FA. Since thioredoxin is known to be the reactive cofactor of ribonucleotid reductase its shortcoming reduces the supply of deoxyribonucleotides thus hindering the DNA and replication repair with resultant chromosomal breaks. Furthermore, depression of tyrosine hydroxylase, the key enzyme of melanine synthesis, could be the basis for the pathognomotic 'café au lait' spots of FA. The observation of thioredoxin reduction in FA cells permits insight into the molecular phathophysiology of FA.

Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture.

PubMed

de Wilde, Adriaan H; Zevenhoven-Dobbe, Jessika C; Beugeling, Corrine; Chatterji, Udayan; de Jong, Danielle; Gallay, Philippe; Szuhai, Karoly; Posthuma, Clara C; Snijder, Eric J

2018-04-01

Cyclophilin A (CypA) is an important host factor in the replication of a variety of RNA viruses. Also the replication of several nidoviruses was reported to depend on CypA, although possibly not to the same extent. These prior studies are difficult to compare, since different nidoviruses, cell lines and experimental set-ups were used. Here, we investigated the CypA dependence of three distantly related nidoviruses that can all replicate in Huh7 cells: the arterivirus equine arteritis virus (EAV), the alphacoronavirus human coronavirus 229E (HCoV-229E), and the betacoronavirus Middle East respiratory syndrome coronavirus (MERS-CoV). The replication of these viruses was compared in the same parental Huh7 cells and in CypA-knockout Huh7 cells generated using CRISPR/Cas9-technology. CypA depletion reduced EAV yields by ~ 3-log, whereas MERS-CoV progeny titers were modestly reduced (3-fold) and HCoV-229E replication was unchanged. This study reveals that the replication of nidoviruses can differ strikingly in its dependence on cellular CypA. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression

PubMed Central

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

2016-01-01

Half of human genome is made 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 (BACs) in Xenopus laevis egg extract. Using this approach we characterized 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 among which 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 inability of 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 on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. PMID:27111843

Class I ADP-Ribosylation Factors Are Involved in Enterovirus 71 Replication

PubMed Central

Wang, Jianmin; Du, Jiang; Jin, Qi

2014-01-01

Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies. PMID:24911624

Rayleigh-Taylor instability in an equal mass plasma

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

Adak, Ashish, E-mail: ashish-adak@yahoo.com; Ghosh, Samiran, E-mail: sran-g@yahoo.com; Chakrabarti, Nikhil, E-mail: nikhil.chakrabarti@saha.ac.in

The Rayleigh-Taylor (RT) instability in an inhomogeneous pair-ion plasma has been analyzed. Considering two fluid model for two species of ions (positive and negative), we obtain the possibility of the existence of RT instability. The growth rate of the RT instability as usual depends on gravity and density gradient scale length. The results are discussed in context of pair-ion plasma experiments.

Initiation of DNA replication requires actin dynamics and formin activity.

PubMed

Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

2017-11-02

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

Phase separation and long-wavelength charge instabilities in spin-orbit coupled systems

NASA Astrophysics Data System (ADS)

Seibold, G.; Bucheli, D.; Caprara, S.; Grilli, M.

2015-01-01

We investigate a two-dimensional electron model with Rashba spin-orbit interaction where the coupling constant g=g(n) depends on the electronic density. It is shown that this dependence may drive the system unstable towards a long-wavelength charge density wave (CDW) where the associated second-order instability occurs in close vicinity to global phase separation. For very low electron densities the CDW instability is nesting-induced and the modulation follows the Fermi momentum kF. At higher density the instability criterion becomes independent of kF and the system may become unstable in a broad momentum range. Finally, upon filling the upper spin-orbit split band, finite momentum instabilities disappear in favor of phase separation alone. We discuss our results with regard to the inhomogeneous phases observed at the LaAlO3/SrTiO3 or LaTiO3/SrTiO3 interfaces.

A higher-order Skyrme model

NASA Astrophysics Data System (ADS)

Gudnason, Sven Bjarke; Nitta, Muneto

2017-09-01

We propose a higher-order Skyrme model with derivative terms of eighth, tenth and twelfth order. Our construction yields simple and easy-to-interpret higher-order Lagrangians. We first show that a Skyrmion with higher-order terms proposed by Marleau has an instability in the form of a baby-Skyrmion string, while the static energies of our construction are positive definite, implying stability against time-independent perturbations. However, we also find that the Hamiltonians of our construction possess two kinds of dynamical instabilities, which may indicate the instability with respect to time-dependent perturbations. Different from the well-known Ostrogradsky instability, the instabilities that we find are intrinsically of nonlinear nature and also due to the fact that even powers of the inverse metric gives a ghost-like higher-order kinetic-like term. The vacuum state is, however, stable. Finally, we show that at sufficiently low energies, our Hamiltonians in the simplest cases, are stable against time-dependent perturbations.

Field Dependence, Perceptual Instability, and Sex Differences.

ERIC Educational Resources Information Center

Bergum, Judith E.; Bergum, Bruce O.

Recent studies have shown perceptual instability to be related to visual creativity as reflected in career choice. In general, those who display greater perceptual instability perceive themselves to be more creative and tend to choose careers related to visual creativity, regardless of their gender. To test the hypothesis that field independents…

Enzymatic Removal of Ribonucleotides from DNA Is Essential for Mammalian Genome Integrity and Development

PubMed Central

Reijns, Martin A.M.; Rabe, Björn; Rigby, Rachel E.; Mill, Pleasantine; Astell, Katy R.; Lettice, Laura A.; Boyle, Shelagh; Leitch, Andrea; Keighren, Margaret; Kilanowski, Fiona; Devenney, Paul S.; Sexton, David; Grimes, Graeme; Holt, Ian J.; Hill, Robert E.; Taylor, Martin S.; Lawson, Kirstie A.; Dorin, Julia R.; Jackson, Andrew P.

2012-01-01

Summary The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells. PMID:22579044

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

PubMed

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-06-15

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

PubMed

Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

2013-08-15

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

PubMed

Pai, Chen-Chun; Kishkevich, Anastasiya; Deegan, Rachel S; Keszthelyi, Andrea; Folkes, Lisa; Kearsey, Stephen E; De León, Nagore; Soriano, Ignacio; de Bruin, Robertus Antonius Maria; Carr, Antony M; Humphrey, Timothy C

2017-09-12

Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

PubMed

Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

2016-05-01

Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

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

PubMed

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.

Replication Rate, Framing, and Format Affect Attitudes and Decisions about Science Claims.

PubMed

Barnes, Ralph M; Tobin, Stephanie J; Johnston, Heather M; MacKenzie, Noah; Taglang, Chelsea M

2016-01-01

A series of five experiments examined how the evaluation of a scientific finding was influenced by information about the number of studies that had successfully replicated the initial finding. The experiments also tested the impact of frame (negative, positive) and numeric format (percentage, natural frequency) on the evaluation of scientific findings. In Experiments 1 through 4, an attitude difference score served as the dependent measure, while a measure of choice served as the dependent measure in Experiment 5. Results from a diverse sample of 188 non-institutionalized U.S. adults (Experiment 2) and 730 undergraduate college students (Experiments 1, 3, and 4) indicated that attitudes became more positive as the replication rate increased and attitudes were more positive when the replication information was framed positively. The results also indicate that the manner in which replication rate was framed had a greater impact on attitude than the replication rate itself. The large effect for frame was attenuated somewhat when information about replication was presented in the form of natural frequencies rather than percentages. A fifth study employing 662 undergraduate college students in a task in which choice served as the dependent measure confirmed the framing effect and replicated the replication rate effect in the positive frame condition, but provided no evidence that the use of natural frequencies diminished the effect.

HTLV-I Tax Increases Genetic Instability by Inducing DNA Double Strand Breaks during DNA Replication and Switching Repair to NHEJ

PubMed Central

Baydoun, Hicham H.; Bai, Xue Tao; Shelton, Shary; Nicot, Christophe

2012-01-01

Background Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood. Results Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway. Conclusions This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation. PMID:22916124

Drosophila Sld5 is essential for normal cell cycle progression and maintenance of genomic integrity

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

Gouge, Catherine A.; Christensen, Tim W., E-mail: christensent@ecu.edu

2010-09-10

Research highlights: {yields} Drosophila Sld5 interacts with Psf1, PPsf2, and Mcm10. {yields} Haploinsufficiency of Sld5 leads to M-phase delay and genomic instability. {yields} Sld5 is also required for normal S phase progression. -- Abstract: Essential for the normal functioning of a cell is the maintenance of genomic integrity. Failure in this process is often catastrophic for the organism, leading to cell death or mis-proliferation. Central to genomic integrity is the faithful replication of DNA during S phase. The GINS complex has recently come to light as a critical player in DNA replication through stabilization of MCM2-7 and Cdc45 as amore » member of the CMG complex which is likely responsible for the processivity of helicase activity during S phase. The GINS complex is made up of 4 members in a 1:1:1:1 ratio: Psf1, Psf2, Psf3, And Sld5. Here we present the first analysis of the function of the Sld5 subunit in a multicellular organism. We show that Drosophila Sld5 interacts with Psf1, Psf2, and Mcm10 and that mutations in Sld5 lead to M and S phase delays with chromosomes exhibiting hallmarks of genomic instability.« less

Electrothermal instability growth in magnetically driven pulsed power liners

NASA Astrophysics Data System (ADS)

Peterson, Kyle J.; Sinars, Daniel B.; Yu, Edmund P.; Herrmann, Mark C.; Cuneo, Michael E.; Slutz, Stephen A.; Smith, Ian C.; Atherton, Briggs W.; Knudson, Marcus D.; Nakhleh, Charles

2012-09-01

This paper explores the role of electro-thermal instabilities on the dynamics of magnetically accelerated implosion systems. Electro-thermal instabilities result from non-uniform heating due to temperature dependence in the conductivity of a material. Comparatively little is known about these types of instabilities compared to the well known Magneto-Rayleigh-Taylor (MRT) instability. We present simulations that show electrothermal instabilities form immediately after the surface material of a conductor melts and can act as a significant seed to subsequent MRT instability growth. We also present the results of several experiments performed on Sandia National Laboratories Z accelerator to investigate signatures of electrothermal instability growth on well characterized initially solid aluminum and copper rods driven with a 20 MA, 100 ns risetime current pulse. These experiments show excellent agreement with electrothermal instability simulations and exhibit larger instability growth than can be explained by MRT theory alone.

Two subunits of human ORC are dispensable for DNA replication and proliferation.

PubMed

Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

2016-12-01

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

Research on aviation fuel instability

NASA Technical Reports Server (NTRS)

Baker, C. E.; Bittker, D. A.; Cohen, S. M.; Seng, G. T.

1983-01-01

The underlying causes of fuel thermal degradation are discussed. Topics covered include: nature of fuel instability and its temperature dependence, methods of measuring the instability, chemical mechanisms involved in deposit formation, and instrumental methods for characterizing fuel deposits. Finally, some preliminary thoughts on design approaches for minimizing the effects of lowered thermal stability are briefly discussed.

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

PubMed Central

Im, Jun-Sub; Park, Soon-Young; Cho, Won-Ho; Bae, Sung-Ho; Hurwitz, Jerard; Lee, Joon-Kyu

2015-01-01

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells. PMID:25602958

Effect of polarization force on the Jeans instability in collisional dusty plasmas

NASA Astrophysics Data System (ADS)

A, ABBASI; M, R. RASHIDIAN VAZIRI

2018-03-01

The Jeans instability in collisional dusty plasmas has been analytically investigated by considering the polarization force effect. Instabilities due to dust-neutral and ion-neutral drags can occur in electrostatic waves of collisional dusty plasmas with self-gravitating particles. In this study, the effect of gravitational force on heavy dust particles is considered in tandem with both the polarization and electrostatic forces. The theoretical framework has been developed and the dispersion relation and instability growth rate have been derived, assuming the plane wave approximation. The derived instability growth rate shows that, in collisional dusty plasmas, the Jeans instability strongly depends on the magnitude of the polarization force.

Self-Replication of Localized Vegetation Patches in Scarce Environments

NASA Astrophysics Data System (ADS)

Bordeu, Ignacio; Clerc, Marcel G.; Couteron, Piere; Lefever, René; Tlidi, Mustapha

2016-09-01

Desertification due to climate change and increasing drought periods is a worldwide problem for both ecology and economy. Our ability to understand how vegetation manages to survive and propagate through arid and semiarid ecosystems may be useful in the development of future strategies to prevent desertification, preserve flora—and fauna within—or even make use of scarce resources soils. In this paper, we study a robust phenomena observed in semi-arid ecosystems, by which localized vegetation patches split in a process called self-replication. Localized patches of vegetation are visible in nature at various spatial scales. Even though they have been described in literature, their growth mechanisms remain largely unexplored. Here, we develop an innovative statistical analysis based on real field observations to show that patches may exhibit deformation and splitting. This growth mechanism is opposite to the desertification since it allows to repopulate territories devoid of vegetation. We investigate these aspects by characterizing quantitatively, with a simple mathematical model, a new class of instabilities that lead to the self-replication phenomenon observed.

Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

PubMed

Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

2016-05-02

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

PubMed

Siede, W; Eckardt, F

1986-01-01

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

Identifying impact of software dependencies on replicability of biomedical workflows.

PubMed

Miksa, Tomasz; Rauber, Andreas; Mina, Eleni

2016-12-01

Complex data driven experiments form the basis of biomedical research. Recent findings warn that the context in which the software is run, that is the infrastructure and the third party dependencies, can have a crucial impact on the final results delivered by a computational experiment. This implies that in order to replicate the same result, not only the same data must be used, but also it must be run on an equivalent software stack. In this paper we present the VFramework that enables assessing replicability of workflows. It identifies whether any differences in software dependencies among two executions of the same workflow exist and whether they have impact on the produced results. We also conduct a case study in which we investigate the impact of software dependencies on replicability of Taverna workflows used in biomedical research of Huntington's disease. We re-execute analysed workflows in environments differing in operating system distribution and configuration. The results show that the VFramework can be used to identify the impact of software dependencies on the replicability of biomedical workflows. Furthermore, we observe that despite the fact that the workflows are executed in a controlled environment, they still depend on specific tools installed in the environment. The context model used by the VFramework improves the deficiencies of provenance traces and documents also such tools. Based on our findings we define guidelines for workflow owners that enable them to improve replicability of their workflows. Copyright © 2016 Elsevier Inc. All rights reserved.

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

PubMed

Rangarajan, Savithri; Woodgate, Roger; Goodman, Myron F

2002-02-01

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.

Observations of a fast transverse instability in the PSR

NASA Astrophysics Data System (ADS)

Neuffer, D.; Colton, E.; Fitzgerald, D.; Hardek, T.; Hutson, R.; Macek, R.; Plum, M.; Thiessen, H.; Wang, T.-S.

1992-09-01

A fast instability with beam loss is observed in the Los Alamos Proton Storage Ring (PSR) when the injected beam current exceeds a threshold value, with both bunched and unbunched beams. Large coherent transverse oscillations occur prior to and during beam loss. The threshold depends strongly on rf voltage, beam-pulse shape, beam size, nonlinear fields, and beam environmental. Results of recent observations of the instability are reported; possible causes of the instability are discussed. Recent measurements and calculations indicate that the instability is an "e-p"-type instability, driven by coupled oscillations with electrons trapped within the proton beam. Future experiments toward further understanding of the instability are discussed, and methods of increasing PSR beam storage are suggested.

Localized instabilities and spinodal decomposition in driven systems in the presence of elasticity

NASA Astrophysics Data System (ADS)

Meca, Esteban; Münch, Andreas; Wagner, Barbara

2018-01-01

We study numerically and analytically the instabilities associated with phase separation in a solid layer on which an external material flux is imposed. The first instability is localized within a boundary layer at the exposed free surface by a process akin to spinodal decomposition. In the limiting static case, when there is no material flux, the coherent spinodal decomposition is recovered. In the present problem, stability analysis of the time-dependent and nonuniform base states as well as numerical simulations of the full governing equations are used to establish the dependence of the wavelength and onset of the instability on parameter settings and its transient nature as the patterns eventually coarsen into a flat moving front. The second instability is related to the Mullins-Sekerka instability in the presence of elasticity and arises at the moving front between the two phases when the flux is reversed. Stability analyses of the full model and the corresponding sharp-interface model are carried out and compared. Our results demonstrate how interface and bulk instabilities can be analyzed within the same framework which allows us to identify and distinguish each of them clearly. The relevance for a detailed understanding of both instabilities and their interconnections in a realistic setting is demonstrated for a system of equations modeling the lithiation and delithiation processes within the context of lithium ion batteries.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites

PubMed Central

Piazza, Aurèle; Adrian, Michael; Samazan, Frédéric; Heddi, Brahim; Hamon, Florian; Serero, Alexandre; Lopes, Judith; Teulade-Fichou, Marie-Paule; Phan, Anh Tuân; Nicolas, Alain

2015-01-01

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or functionin vivo are unknown. InS. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with thein vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved inC. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequencesin vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to formin vivo. PMID:25956747

Mutational Analysis of Mismatch Repair Genes, hMLH1 and hMSH2, in Sporadic Endometrial Carcinomas with Microsatellite Instability

PubMed Central

Kobayashi, Kanji; Matsushima, Mieko; Koi, Sumiko; Saito, Hiroko; Sagae, Satoru; Kudo, Ryuichi

1996-01-01

Microsatellite instability, monitored by replication error (RER), bas been observed in both sporadic and hereditary types of endometrial carcinoma. In the hereditary tumors, this instability is considered to be caused by a germline defect in the DNA mismatch‐repair system. We previously reported that nearly one‐quarter of sporadic endometrial carcinomas examined revealed an RER‐positive phenotype at multiple microsatellite loci. To investigate the role of genetic alterations of DNA mismatch‐repair genes in sporadic endometrial carcinomas, we screened 18 RER(+) endometrial carcinomas for mutations of hMLH1 and hMSH2. Although we found no germline mutations, we detected two somatic mutations of hMLH1 in a single endometrial cancer; these two mutations had occurred on different alleles, suggesting that two separate mutational events had affected both copies of hMLH1 in this particular tumor. These data implied that mutations of hMLH1 or hMSH2 play limited roles in the development of sporadic endometrial carcinomas, and that the tumors with genetic instability might have alterations of other mismatch‐repair genes, such as hPMS1 and hPMS2, or of unknown genes related to the mismatch‐repair system. PMID:8609062

Human embryonic stem cells fail to activate CHK1 and commit to apoptosis in response to DNA replication stress.

PubMed

Desmarais, Joëlle A; Hoffmann, Michele J; Bingham, Gregg; Gagou, Mary E; Meuth, Mark; Andrews, Peter W

2012-07-01

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, γH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability. Copyright © 2012 AlphaMed Press.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome.

PubMed

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-09-30

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts' fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates 'mother bias' (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome

PubMed Central

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-01-01

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts’ fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates ‘mother bias’ (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. PMID:27492289

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

PubMed Central

Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

2013-01-01

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity. PMID:23564203

DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

PubMed

Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

2013-06-01

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

Replication Rate, Framing, and Format Affect Attitudes and Decisions about Science Claims

PubMed Central

Barnes, Ralph M.; Tobin, Stephanie J.; Johnston, Heather M.; MacKenzie, Noah; Taglang, Chelsea M.

2016-01-01

A series of five experiments examined how the evaluation of a scientific finding was influenced by information about the number of studies that had successfully replicated the initial finding. The experiments also tested the impact of frame (negative, positive) and numeric format (percentage, natural frequency) on the evaluation of scientific findings. In Experiments 1 through 4, an attitude difference score served as the dependent measure, while a measure of choice served as the dependent measure in Experiment 5. Results from a diverse sample of 188 non-institutionalized U.S. adults (Experiment 2) and 730 undergraduate college students (Experiments 1, 3, and 4) indicated that attitudes became more positive as the replication rate increased and attitudes were more positive when the replication information was framed positively. The results also indicate that the manner in which replication rate was framed had a greater impact on attitude than the replication rate itself. The large effect for frame was attenuated somewhat when information about replication was presented in the form of natural frequencies rather than percentages. A fifth study employing 662 undergraduate college students in a task in which choice served as the dependent measure confirmed the framing effect and replicated the replication rate effect in the positive frame condition, but provided no evidence that the use of natural frequencies diminished the effect. PMID:27920743

The Midblastula Transition Defines the Onset of Y RNA-Dependent DNA Replication in Xenopus laevis ▿

PubMed Central

Collart, Clara; Christov, Christo P.; Smith, James C.; Krude, Torsten

2011-01-01

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery. PMID:21791613

On the instability of a liquid sheet moving in vacuum

NASA Astrophysics Data System (ADS)

Sisoev, G. M.; Osiptsov, A. N.; Koroteev, A. A.

2018-03-01

A linear stability analysis of a non-isothermal liquid sheet moving in vacuum is studied taking into account the temperature dependencies of the liquid viscosity, thermal conductivity, and surface tension coefficients. It is found that there are two mechanisms of instability. The short-wave instability is caused by viscosity stratification across the sheet due to nonuniform temperature profiles developed downstream in the cooling sheet. The long-wave thermocapillary instability is caused by the temperature gradient along the sheet surfaces. Computed examples of steady flows and their instabilities demonstrated that the unstable short waves have much larger amplification factors.

Evaporation effects in a shock-driven multiphase instability with a spherical interface

NASA Astrophysics Data System (ADS)

Paudel, Manoj; Dahal, Jeevan; McFarland, Jacob

2017-11-01

This talk presents results from 3D numerical simulations of a shock driven instability of a gas-particle system with a spherical interface. Two cases, one with an evaporating particle cloud and another with a gas only approximation of this particle cloud, were run in the hydrodynamics code FLASH, developed at University of Chicago. It is shown that the gas only approximation, a classical Richtmyer Meshkov instability, cannot replicate effects from particles like, lag, clustering, and evaporation. Instead, both gas hydrodynamics and particle properties influence one another and are coupled. Results are presented to highlight the coupling of interface evolution and particle evaporation. Qualitative and quantitative differences in the RMI and SDMI are presented by studying the change in gas properties like density and vorticity within the interface. Similarly, the effect of gas hydrodynamics on particles distribution and evaporation is studied. Particle evaporation rates are compared with 1D models and show poor agreement. The variation in evaporation rates for similar sized particles and the role of gas hydrodynamics in these variation is explored.

Pattern formation, social forces, and diffusion instability in games with success-driven motion

NASA Astrophysics Data System (ADS)

Helbing, Dirk

2009-02-01

A local agglomeration of cooperators can support the survival or spreading of cooperation, even when cooperation is predicted to die out according to the replicator equation, which is often used in evolutionary game theory to study the spreading and disappearance of strategies. In this paper, it is shown that success-driven motion can trigger such local agglomeration and may, therefore, be used to supplement other mechanisms supporting cooperation, like reputation or punishment. Success-driven motion is formulated here as a function of the game-theoretical payoffs. It can change the outcome and dynamics of spatial games dramatically, in particular as it causes attractive or repulsive interaction forces. These forces act when the spatial distributions of strategies are inhomogeneous. However, even when starting with homogeneous initial conditions, small perturbations can trigger large inhomogeneities by a pattern-formation instability, when certain conditions are fulfilled. Here, these instability conditions are studied for the prisoner’s dilemma and the snowdrift game. Furthermore, it is demonstrated that asymmetrical diffusion can drive social, economic, and biological systems into the unstable regime, if these would be stable without diffusion.

Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Inducible Factor-Dependent Extension of the Replicative Life Span during Hypoxia▿

PubMed Central

Bell, Eric L.; Klimova, Tatyana A.; Eisenbart, James; Schumacker, Paul T.; Chandel, Navdeep S.

2007-01-01

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span. PMID:17562866

Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

PubMed Central

Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.

2013-01-01

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298

Emotionally up and down, behaviorally to and fro: Drinking motives mediate the synergistic effects of urgency and emotional instability on alcohol outcomes

PubMed Central

Dvorak, Robert D.; Kuvaas, Nicholas J.; Lamis, Dorian A.; Pearson, Matthew R.; Stevenson, Brittany L.

2017-01-01

Emotional and behavioral regulation has been linked to coping and enhancement motives and associated with different patterns of alcohol use and problems. The current studies examined emotional instability, urgency, and internal drinking motives as predictors of alcohol dependence symptoms and DSM-5 Alcohol Use Disorder. In Study 1, college drinkers (n = 621) completed alcohol involvement and behavioral/emotional functioning assessments. There was an indirect association between emotional instability and dependence symptoms via both coping and enhancement drinking motives which was potentiated by trait urgency. In Study 2, college drinkers (n = 510) completed alcohol involvement, behavioral/emotional functioning, and AUD criteria assessments. A significant indirect effect from emotional instability to the likelihood of meeting AUD criteria, via drinking to cope was found, again potentiated by urgency. PMID:27075609

A minimization principle for the description of modes associated with finite-time instabilities

PubMed Central

Babaee, H.

2016-01-01

We introduce a minimization formulation for the determination of a finite-dimensional, time-dependent, orthonormal basis that captures directions of the phase space associated with transient instabilities. While these instabilities have finite lifetime, they can play a crucial role either by altering the system dynamics through the activation of other instabilities or by creating sudden nonlinear energy transfers that lead to extreme responses. However, their essentially transient character makes their description a particularly challenging task. We develop a minimization framework that focuses on the optimal approximation of the system dynamics in the neighbourhood of the system state. This minimization formulation results in differential equations that evolve a time-dependent basis so that it optimally approximates the most unstable directions. We demonstrate the capability of the method for two families of problems: (i) linear systems, including the advection–diffusion operator in a strongly non-normal regime as well as the Orr–Sommerfeld/Squire operator, and (ii) nonlinear problems, including a low-dimensional system with transient instabilities and the vertical jet in cross-flow. We demonstrate that the time-dependent subspace captures the strongly transient non-normal energy growth (in the short-time regime), while for longer times the modes capture the expected asymptotic behaviour. PMID:27118900

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

PubMed

Liu, Xiao; Dang, Yunkun; Matsu-Ura, Toru; He, Yubo; He, Qun; Hong, Christian I; Liu, Yi

2017-07-20

Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism. Published by Elsevier Inc.

Tumors overexpressing RNF168 show altered DNA repair and responses to genotoxic treatments, genomic instability and resistance to proteotoxic stress.

PubMed

Chroma, K; Mistrik, M; Moudry, P; Gursky, J; Liptay, M; Strauss, R; Skrott, Z; Vrtel, R; Bartkova, J; Kramara, J; Bartek, J

2017-04-27

Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.

Sequential replication-coupled destruction at G1/S ensures genome stability

PubMed Central

Coleman, Kate E.; Grant, Gavin D.; Haggerty, Rachel A.; Brantley, Kristen; Shibata, Etsuko; Workman, Benjamin D.; Dutta, Anindya; Varma, Dileep; Purvis, Jeremy E.; Cook, Jeanette Gowen

2015-01-01

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4Cdt2, which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4Cdt2 targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNADNA) and recruits CRL4Cdt2. Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNADNA. Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability. PMID:26272819

Disintegration of Nascent Replication Bubbles during Thymine Starvation Triggers RecA- and RecBCD-dependent Replication Origin Destruction*

PubMed Central

Kuong, Kawai J.; Kuzminov, Andrei

2012-01-01

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA+ cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction. PMID:22621921

Thermal effects in rapid directional solidification - Linear theory

NASA Technical Reports Server (NTRS)

Huntley, D. A.; Davis, S. H.

1993-01-01

We study the morphological instability of the planar solid/liquid interface for a unidirectionally-solidified dilute binary mixture. We use a model developed by Boettinger et al. (1985, 1986), Aziz (1982), and Jackson et al. (1980), which allows for nonequilibrium effects on the interface through velocity-dependent segregation and attachment kinetics. Two types of instabilities are found in the linear stability analysis: (1) a cellular instability, and (2) an oscillatory instability driven by disequilibrium effects. Merchant and Davis (1990) characterized these instabilities subject to the frozen-temperature approximation (FTA). The present work relaxes the FTA by including the effects of latent heat and the full temperature distribution. Thermal effects slightly postpone the onset of the cellular instability but dramatically postpone the onset of the oscillatory instability; however, the absolute-stability conditions, at which at high speed the cellular and oscillatory instabilities are suppressed, remain unchanged from the FTA.

Transcription as a Threat to Genome Integrity.

PubMed

Gaillard, Hélène; Aguilera, Andrés

2016-06-02

Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant.

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation.

PubMed

Sansam, Courtney G; Goins, Duane; Siefert, Joseph C; Clowdus, Emily A; Sansam, Christopher L

2015-03-01

S-phase cyclin-dependent kinases (CDKs) stimulate replication initiation and accelerate progression through the replication timing program, but it is unknown which CDK substrates are responsible for these effects. CDK phosphorylation of the replication factor TICRR (TopBP1-interacting checkpoint and replication regulator)/TRESLIN is required for DNA replication. We show here that phosphorylated TICRR is limiting for S-phase progression. Overexpression of a TICRR mutant with phosphomimetic mutations at two key CDK-phosphorylated residues (TICRR(TESE)) stimulates DNA synthesis and shortens S phase by increasing replication initiation. This effect requires the TICRR region that is necessary for its interaction with MDM two-binding protein. Expression of TICRR(TESE) does not grossly alter the spatial organization of replication forks in the nucleus but does increase replication clusters and the number of replication forks within each cluster. In contrast to CDK hyperactivation, the acceleration of S-phase progression by TICRR(TESE) does not induce DNA damage. These results show that CDK can stimulate initiation and compress the replication timing program by phosphorylating a single protein, suggesting a simple mechanism by which S-phase length is controlled. © 2015 Sansam et al.; Published by Cold Spring Harbor Laboratory Press.

Parametric instabilities of finite-amplitude, circularly polarized Alfven waves in an anisotropic plasma

NASA Technical Reports Server (NTRS)

Hamabata, Hiromitsu

1993-01-01

A class of parametric instabilities of finite-amplitude, circularly polarized Alfven waves in a plasma with pressure anisotropy is studied by application of the CGL equations. A linear perturbation analysis is used to find the dispersion relation governing the instabilities, which is a fifth-order polynomial and is solved numerically. A large-amplitude, circularly polarized wave is unstable with respect to decay into three waves: one sound-like wave and two side-band Alfven-like waves. It is found that, in addition to the decay instability, two new instabilities that are absent in the framework of the MHD equations can occur, depending on the plasma parameters.

Tumor Suppression by BRCA-1: A Critical Role at DNA Replication Forks

DTIC Science & Technology

2006-10-01

replication defect. We wished to test the hypothesis that BRCA1/BARD1 function during DNA replication supporting DNA transactions at replication forks. We...are using cell-free extracts derived from Xenopus laevis eggs that support: 1. Semi-conservative, cell-cycle regulated DNA replication ; 2. Many facets...complex assembles to chromatin in a DNA replication -dependent manner. Finally, we show that BRCA1/BARD1 loading to chromatin does not dramatically

Temporary employment and health: a review.

PubMed

Virtanen, Marianna; Kivimäki, Mika; Joensuu, Matti; Virtanen, Pekka; Elovainio, Marko; Vahtera, Jussi

2005-06-01

We aimed to review evidence on the relationship between temporary employment and health, and to see whether the association is dependent on outcome measure, instability of employment, and contextual factors. We systematically searched for studies of temporary employment and various health outcomes and critically appraised 27 studies. The review suggests higher psychological morbidity among temporary workers compared with permanent employees. According to some studies, temporary workers also have a higher risk of occupational injuries but their sickness absence is lower. Morbidity may be higher in temporary jobs with high employment instability and in countries with a lower number of temporary workers and unemployed workers. The evidence indicates an association between temporary employment and psychological morbidity. The health risk may depend on instability of temporary employment and the context. Confounding by occupation may have biased some of the studies. Additional research to clarify the role of employment instability, hazard accumulation, and selection is recommended.

Multi-water-bag models of ion temperature gradient instability in cylindrical geometry

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

Coulette, David; Besse, Nicolas

2013-05-15

Ion temperature gradient instabilities play a major role in the understanding of anomalous transport in core fusion plasmas. In the considered cylindrical geometry, ion dynamics is described using a drift-kinetic multi-water-bag model for the parallel velocity dependency of the ion distribution function. In a first stage, global linear stability analysis is performed. From the obtained normal modes, parametric dependencies of the main spectral characteristics of the instability are then examined. Comparison of the multi-water-bag results with a reference continuous Maxwellian case allows us to evaluate the effects of discrete parallel velocity sampling induced by the Multi-Water-Bag model. Differences between themore » global model and local models considered in previous works are discussed. Using results from linear, quasilinear, and nonlinear numerical simulations, an analysis of the first stage saturation dynamics of the instability is proposed, where the divergence between the three models is examined.« less

Inhibition of polyomavirus ori-dependent DNA replication by mSin3B.

PubMed

Xie, An-Yong; Folk, William R

2002-12-01

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) ori-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.

Genetic instability in urinary bladder cancer: An evolving hallmark.

PubMed

Wadhwa, N; Mathew, B B; Jatawa, S K; Tiwari, A

2013-01-01

Bladder cancer is a major health-care concern. A successful treatment of bladder cancer depends on its early diagnosis at the initial stage. Genetic instability is an essential early step toward the development of bladder cancer. This instability is found more often at the chromosomal level than at the nucleotide level. Microsatellite and chromosomal instability markers can be used as a prognostic marker for screening bladder cancer. Bladder cancer can be distinguished in two different categories according to genetic instability: Cancers with chromosomal level instability and cancers with nucleotide level instability. Deoxyribonucleic acid (DNA) mismatch repair (MMR) system and its correlation with other biologic pathway, both are essential to understand the basic mechanisms of cancer development. Microsatellite instability occurs due to defects in DNA MMR genes, including human mutL homolog 1 and human mutL homolog 2. Chromosomal alterations including deletions on chromosome 3, 8, 9, 11, 13, 17 have been detected in bladder cancer. In the current review, the most recent literature of genetic instability in urinary bladder cancer has been summarized.

Transcription as a source of genome instability

PubMed Central

Kim, Nayun; Jinks-Robertson, Sue

2012-01-01

Alterations in genome sequence and structure contribute to somatic disease, affect the fitness of subsequent generations and drive evolutionary processes. The critical roles of highly accurate replication and efficient repair in maintaining overall genome integrity are well known, but the more localized stability costs associated with transcribing DNA into RNA molecules are less appreciated. Here we review the diverse ways that the essential process of transcription alters the underlying DNA template and thereby modifies the genetic landscape. PMID:22330764

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

PubMed Central

Schwab, Rebekka A.V.; Niedzwiedz, Wojciech

2011-01-01

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope. PMID:22064662

Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells.

PubMed

Bursomanno, Sara; Beli, Petra; Khan, Asif M; Minocherhomji, Sheroy; Wagner, Sebastian A; Bekker-Jensen, Simon; Mailand, Niels; Choudhary, Chunaram; Hickson, Ian D; Liu, Ying

2015-01-01

SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational repair. We have also shown that deficiency of POLD3 leads to an increase in RPA-bound ssDNA when cells are under replication stress, suggesting that POLD3 plays a role in the cellular response to DNA replication stress. Considering that DNA replication stress is a source of genome instability, and that excessive replication stress is a hallmark of pre-neoplastic and tumor cells, our characterization of SUMO2 targets during a perturbed S-phase should provide a valuable resource for future functional studies in the fields of DNA metabolism and cancer biology. Copyright © 2014 Elsevier B.V. All rights reserved.

Human stem cell–derived astrocytes replicate human prions in a PRNP genotype–dependent manner

PubMed Central

Krejciova, Zuzana; Alibhai, James; Zhao, Chen; Rzechorzek, Nina M.; Ullian, Erik M.; Manson, Jean

2017-01-01

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery. PMID:29141869

Suppression of the DHX9 Helicase Induces Premature Senescence in Human Diploid Fibroblasts in a p53-dependent Manner*

PubMed Central

Lee, Teresa; Di Paola, Domenic; Malina, Abba; Mills, John R.; Kreps, Amina; Grosse, Frank; Tang, Hengli; Zannis-Hadjopoulos, Maria; Larsson, Ola; Pelletier, Jerry

2014-01-01

DHX9 is an ATP-dependent DEXH box helicase with a multitude of cellular functions. Its ability to unwind both DNA and RNA, as well as aberrant, noncanonical polynucleotide structures, has implicated it in transcriptional and translational regulation, DNA replication and repair, and maintenance of genome stability. We report that loss of DHX9 in primary human fibroblasts results in premature senescence, a state of irreversible growth arrest. This is accompanied by morphological defects, elevation of senescence-associated β-galactosidase levels, and changes in gene expression closely resembling those encountered during replicative (telomere-dependent) senescence. Activation of the p53 signaling pathway was found to be essential to this process. ChIP analysis and investigation of nascent DNA levels revealed that DHX9 is associated with origins of replication and that its suppression leads to a reduction of DNA replication. Our results demonstrate an essential role of DHX9 in DNA replication and normal cell cycle progression. PMID:24990949

Rapid DNA Synthesis During Early Drosophila Embryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function.

PubMed

Lesly, Shera; Bandura, Jennifer L; Calvi, Brian R

2017-11-01

Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNA replication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty ( hd ), and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele, hd 272-9 , were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects of thin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification and eggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd 272-9 allele, resulting in reduced maternal Hd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosome bridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the result of a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein is essential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd 272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularly sensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in the human ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms. Copyright © 2017 by the Genetics Society of America.

Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest.

PubMed

Kehrli, Keffy; Phelps, Michael; Lazarchuk, Pavlo; Chen, Eleanor; Monnat, Ray; Sidorova, Julia M

2016-11-18

The WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN- or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1-3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Investigation of instability of displacement front in non-isothermal flow problems

NASA Astrophysics Data System (ADS)

Syulyukina, Natalia; Pergament, Anna

2012-11-01

In this paper, we investigate the issues of front instability arising in non-isothermal flow displacement processes. The problem of two-phase flow of immiscible fluids, oil and water, is considered, including sources and dependence of viscosity on temperature. Three-dimensional problem with perturbation close to the injection well was considered to find the characteristic scale of the instability. As a result of numerical calculations, theoretical studies on the development of the instability due to the fact that the viscosity of the displacing fluid is less than the viscosity of the displaced have been confirmed. The influence of temperature on the evolution of the instability was considered. For this purpose, the dependence of oil viscosity on temperature has been added to the problem. Numerical calculations were carried out for different values of temperature and it was shown that with increasing of production rate. Thus, it has been demonstrated that the selection of the optimal temperature for injected fluids a possible way for stimulation of oil production also delaying the field water-flooding. This work was supporting by the RFBR grant 12-01-00793-a.

Global instability in a laminar boundary layer perturbed by an isolated roughness element

NASA Astrophysics Data System (ADS)

Puckert, Dominik K.; Rist, Ulrich

2018-03-01

Roughness-induced boundary-layer instabilities are investigated by means of hot-film anemometry in a water channel to provide experimental evidence of a global instability. It is shown that the roughness wake dynamics depends on extrinsic disturbances (amplifier) at subcritical Reynolds numbers, whereas intrinsic, self-sustained oscillations (wavemaker) are suspected at supercritical Reynolds numbers. The critical Reynolds number, therefore, separates between two different instability mechanisms. Furthermore, the critical Reynolds number from recent theoretical results is successfully confirmed in this experiment, supporting the physical relevance of 3-d global stability theory.

Geoid Anomalies and Dynamic Topography from Time Dependent, Spherical Axisymmetric Mantle Convection

NASA Technical Reports Server (NTRS)

Kiefer, Walter S.; Kellogg, Louise H.

1998-01-01

Geoid anomalies and dynamic topography are two important diagnostics of mantle convection. We present geoid and topography results for several time-dependent convection models in spherical axisymmetric geometry for Rayleigh numbers between 10(exp 6) and 10(exp 7) with depth-dependent viscosity and mixtures of bottom and internal heating. The models are strongly chaotic, with boundary layer instabilities erupting out of both thermal boundary layers. In some instances, instabilities from one boundary layer influence the development of instabilities in the other boundary layer. Such coupling between events at the top and bottom of the mantle has been suggested to play a role in a mid-Cretaceous episode of enhanced volcanism in the Pacific. These boundary layer instabilities produce large temporal variations in the geoid anomalies and dynamic nd to the topography associated with the convection. The amplitudes of these fluctuations depend on the detailed model parameter,.% it of this but fluctuations of 30-50% relative to the time-averaged geoid and topography are common. The convective planform is strongly sensitive to the specific initial conditions. Convection cells with larger aspect ratio tend to have larger fractional fluctuations in their geoid and topography amplitudes, because boundary layer instabilities have more time to develop in long cells. In some instances, we observe low-amplitude topographic highs adjacent to the topographic lows produced by cold downwellings. We discuss applications of these results to several situations, including the temporal variability of m basis. hotspots such as Hawaii, the topography of subduction zone outer rises, and the topography of coronae on Venus.

Two interferon-independent double-stranded RNA-induced host defense strategies suppress the common cold virus at warm temperature.

PubMed

Foxman, Ellen F; Storer, James A; Vanaja, Kiran; Levchenko, Andre; Iwasaki, Akiko

2016-07-26

Most strains of rhinovirus (RV), the common cold virus, replicate better at cool temperatures found in the nasal cavity (33-35 °C) than at lung temperature (37 °C). Recent studies found that although 37 °C temperature suppressed RV growth largely by engaging the type 1 IFN response in infected epithelial cells, a significant temperature dependence to viral replication remained in cells devoid of IFN induction or signaling. To gain insight into IFN-independent mechanisms limiting RV replication at 37 °C, we studied RV infection in human bronchial epithelial cells and H1-HeLa cells. During the single replication cycle, RV exhibited temperature-dependent replication in both cell types in the absence of IFN induction. At 37 °C, earlier signs of apoptosis in RV-infected cells were accompanied by reduced virus production. Furthermore, apoptosis of epithelial cells was enhanced at 37 °C in response to diverse stimuli. Dynamic mathematical modeling and B cell lymphoma 2 (BCL2) overexpression revealed that temperature-dependent host cell death could partially account for the temperature-dependent growth observed during RV amplification, but also suggested additional mechanisms of virus control. In search of a redundant antiviral pathway, we identified a role for the RNA-degrading enzyme RNAseL. Simultaneous antagonism of apoptosis and RNAseL increased viral replication and dramatically reduced temperature dependence. These findings reveal two IFN-independent mechanisms active in innate defense against RV, and demonstrate that even in the absence of IFNs, temperature-dependent RV amplification is largely a result of host cell antiviral restriction mechanisms operating more effectively at 37 °C than at 33 °C.

DNA Double-Strand Break Repair as Determinant of Cellular Radiosensitivity to Killing and Target in Radiation Therapy

PubMed Central

Mladenov, Emil; Magin, Simon; Soni, Aashish; Iliakis, George

2013-01-01

Radiation therapy plays an important role in the management of a wide range of cancers. Besides innovations in the physical application of radiation dose, radiation therapy is likely to benefit from novel approaches exploiting differences in radiation response between normal and tumor cells. While ionizing radiation induces a variety of DNA lesions, including base damages and single-strand breaks, the DNA double-strand break (DSB) is widely considered as the lesion responsible not only for the aimed cell killing of tumor cells, but also for the general genomic instability that leads to the development of secondary cancers among normal cells. Homologous recombination repair (HRR), non-homologous end-joining (NHEJ), and alternative NHEJ, operating as a backup, are the major pathways utilized by cells for the processing of DSBs. Therefore, their function represents a major mechanism of radiation resistance in tumor cells. HRR is also required to overcome replication stress – a potent contributor to genomic instability that fuels cancer development. HRR and alternative NHEJ show strong cell-cycle dependency and are likely to benefit from radiation therapy mediated redistribution of tumor cells throughout the cell-cycle. Moreover, the synthetic lethality phenotype documented between HRR deficiency and PARP inhibition has opened new avenues for targeted therapies. These observations make HRR a particularly intriguing target for treatments aiming to improve the efficacy of radiation therapy. Here, we briefly describe the major pathways of DSB repair and review their possible contribution to cancer cell radioresistance. Finally, we discuss promising alternatives for targeting DSB repair to improve radiation therapy and cancer treatment. PMID:23675572

The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription▿

PubMed Central

Dutta, Chaitali; Patel, Prasanta K.; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

2008-01-01

The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes. PMID:18662996

The activities of eukaryotic replication origins in chromatin.

PubMed

Weinreich, Michael; Palacios DeBeer, Madeleine A; Fox, Catherine A

2004-03-15

DNA replication initiates at chromosomal positions called replication origins. This review will focus on the activity, regulation and roles of replication origins in Saccharomyces cerevisiae. All eukaryotic cells, including S. cerevisiae, depend on the initiation (activity) of hundreds of replication origins during a single cell cycle for the duplication of their genomes. However, not all origins are identical. For example, there is a temporal order to origin activation with some origins firing early during the S-phase and some origins firing later. Recent studies provide evidence that posttranslational chromatin modifications, heterochromatin-binding proteins and nucleosome positioning can control the efficiency and/or timing of chromosomal origin activity in yeast. Many more origins exist than are necessary for efficient replication. The availability of excess replication origins leaves individual origins free to evolve distinct forms of regulation and/or roles in chromosomes beyond their fundamental role in DNA synthesis. We propose that some origins have acquired roles in controlling chromatin structure and/or gene expression. These roles are not linked obligatorily to replication origin activity per se, but instead exploit multi-subunit replication proteins with the potential to form context-dependent protein-protein interactions.

Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

PubMed

Bellelli, Roberto; Borel, Valerie; Logan, Clare; Svendsen, Jennifer; Cox, Danielle E; Nye, Emma; Metcalfe, Kay; O'Connell, Susan M; Stamp, Gordon; Flynn, Helen R; Snijders, Ambrosius P; Lassailly, François; Jackson, Andrew; Boulton, Simon J

2018-05-17

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4 -/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4 -/- p53 +/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Self-Replication of Localized Vegetation Patches in Scarce Environments

PubMed Central

Bordeu, Ignacio; Clerc, Marcel G.; Couteron, Piere; Lefever, René; Tlidi, Mustapha

2016-01-01

Desertification due to climate change and increasing drought periods is a worldwide problem for both ecology and economy. Our ability to understand how vegetation manages to survive and propagate through arid and semiarid ecosystems may be useful in the development of future strategies to prevent desertification, preserve flora—and fauna within—or even make use of scarce resources soils. In this paper, we study a robust phenomena observed in semi-arid ecosystems, by which localized vegetation patches split in a process called self-replication. Localized patches of vegetation are visible in nature at various spatial scales. Even though they have been described in literature, their growth mechanisms remain largely unexplored. Here, we develop an innovative statistical analysis based on real field observations to show that patches may exhibit deformation and splitting. This growth mechanism is opposite to the desertification since it allows to repopulate territories devoid of vegetation. We investigate these aspects by characterizing quantitatively, with a simple mathematical model, a new class of instabilities that lead to the self-replication phenomenon observed. PMID:27650430

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

PubMed

Feeney, Laura; Muñoz, Ivan M; Lachaud, Christophe; Toth, Rachel; Appleton, Paul L; Schindler, Detlev; Rouse, John

2017-06-01

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA.

PubMed

Lopez, Christopher R; Singh, Shivani; Hambarde, Shashank; Griffin, Wezley C; Gao, Jun; Chib, Shubeena; Yu, Yang; Ira, Grzegorz; Raney, Kevin D; Kim, Nayun

2017-06-02

G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A 4-week neuromuscular training program and gait patterns at the ankle joint.

PubMed

Coughlan, Garrett; Caulfield, Brian

2007-01-01

Previous research into the rehabilitation of ankle sprains has primarily focused on outcome measures that do not replicate functional activities, thus making it difficult to extrapolate the results relative to the weight-bearing conditions under which most ankle sprains occur. To measure the effects of a training program on gait during walking and running in an active athletic population. Matched-pairs, controlled trial. University motion analysis laboratory. Ten subjects from an athletic population (7 healthy, 3 with functional ankle instability: age = 25.8 +/- 3.9 years, height = 177.6 +/- 6.1 cm, mass = 66.8 +/- 7.4 kg) and 10 controls matched for age, sex, activity, and ankle instability (7 healthy, 3 with functional ankle instability: age = 27.4 +/- 5.8 years, height = 178.7 +/- 10.8 cm, mass = 71.6 +/- 10.0 kg). A 4-week neuromuscular training program undertaken by the treatment group. We measured ankle position and velocity in the frontal (x) and sagittal (y) planes in all subjects during treadmill walking and running for the periods 100 milliseconds before heel strike, at heel strike, and 100 milliseconds after heel strike. A 4-week neuromuscular training program resulted in no significant changes in ankle position or velocity during treadmill walking and running. The mechanisms by which neuromuscular training improves function in normal subjects and those with functional ankle instability do not appear to result in measurable changes in gait kinematics. Our findings raise issues regarding methods of ankle sprain rehabilitation and the measurement of their effectiveness in improving functional activities. Further research in a larger population with functional ankle instability is necessary.

On the instability of wave-fields with JONSWAP spectra to inhomogeneous disturbances, and the consequent long-time evolution

NASA Astrophysics Data System (ADS)

Ribal, A.; Stiassnie, M.; Babanin, A.; Young, I.

2012-04-01

The instability of two-dimensional wave-fields and its subsequent evolution in time are studied by means of the Alber equation for narrow-banded random surface-waves in deep water subject to inhomogeneous disturbances. A linear partial differential equation (PDE) is obtained after applying an inhomogeneous disturbance to the Alber's equation and based on the solution of this PDE, the instability of the ocean wave surface is studied for a JONSWAP spectrum, which is a realistic ocean spectrum with variable directional spreading and steepness. The steepness of the JONSWAP spectrum depends on γ and α which are the peak-enhancement factor and energy scale of the spectrum respectively and it is found that instability depends on the directional spreading, α and γ. Specifically, if the instability stops due to the directional spreading, increase of the steepness by increasing α or γ can reactivate it. This result is in qualitative agreement with the recent large-scale experiment and new theoretical results. In the instability area of α-γ plane, a long-time evolution has been simulated by integrating Alber's equation numerically and recurrent evolution is obtained which is the stochastic counterpart of the Fermi-Pasta-Ulam recurrence obtained for the cubic Schrödinger equation.

Instability growth for magnetized liner inertial fusion seeded by electro-thermal, electro-choric, and material strength effects

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

Pecover, J. D.; Chittenden, J. P.

A critical limitation of magnetically imploded systems such as magnetized liner inertial fusion (MagLIF) [Slutz et al., Phys. Plasmas 17, 056303 (2010)] is the magneto-Rayleigh-Taylor (MRT) instability which primarily disrupts the outer surface of the liner. MagLIF-relevant experiments have showed large amplitude multi-mode MRT instability growth growing from surface roughness [McBride et al., Phys. Rev. Lett. 109, 135004 (2012)], which is only reproduced by 3D simulations using our MHD code Gorgon when an artificially azimuthally correlated initialisation is added. We have shown that the missing azimuthal correlation could be provided by a combination of the electro-thermal instability (ETI) and anmore » “electro-choric” instability (ECI); describing, respectively, the tendency of current to correlate azimuthally early in time due to temperature dependent Ohmic heating; and an amplification of the ETI driven by density dependent resistivity around vapourisation. We developed and implemented a material strength model in Gorgon to improve simulation of the solid phase of liner implosions which, when applied to simulations exhibiting the ETI and ECI, gave a significant increase in wavelength and amplitude. Full circumference simulations of the MRT instability provided a significant improvement on previous randomly initialised results and approached agreement with experiment.« less

Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication.

PubMed

Meier, Jeffery L; Keller, Michael J; McCoy, James J

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer's orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at -300 or -345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

Magnetohydrodynamic Simulations of the Wiggle Instability in Spiral Galaxies

NASA Astrophysics Data System (ADS)

Tanaka, Minoru; Wada, Keiichi; Machida, Mami; Matsumoto, Ryoji; Miyaji, Shigeki

2005-09-01

We studied the stability of galactic spiral shocks through two dimensional global magnetohydrodynamic simulations. Recently, Wada & Koda (2003) showed, using global hydrodynamic simulations, that galactic gas flows behind a spiral shock becomes unstable against a perturbation parallel to the shock front and form spur-like density structures. They attributed the origin of this wiggle instability to the Kelvin-Helmholtz (K-H) instability triggered by the acceleration of the gas behind the shock. We carried out global simulations including galactic magnetic fields. The initial magnetic field is assumed to be either uniform or purely toroidal. We found that although the magnetic field reduces the growth rate of the K-H instability, wiggle instability develops even in galaxies with μG magnetic fields. We also present the results of local simulations to demonstrate the dependence of the growth rate of the instability with the wavelength. The interval of spurs is determined by the most unstable wavelength of the wiggle instability.

Spectrum of magnetic resonance imaging findings in clinical glenohumeral instability

PubMed Central

Jana, Manisha; Srivastava, Deep Narayan; Sharma, Raju; Gamanagatti, Shivanand; Nag, Hiralal; Mittal, Ravi; Upadhyay, Ashish Dutt

2011-01-01

The glenohumeral joint is the most commonly dislocated joint in the body, and anterior instability is the most common type of shoulder instability. Depending on the etiology and the age of the patient, there may be associated injuries, for example, to the anterior-inferior labro-ligamentous structures (in young individuals with traumatic instability) or to the bony components (commoner in the elderly), which are best visualized using MRI and MR arthrography. Anterior instability is associated with a Bankart lesion and its variants and abnormalities of the anterior band of the inferior glenohumeral ligament (IGHL), whereas posterior instability is associated with reverse Bankart and reverse Hill-Sachs lesions. Cases of multidirectional instability often have no labral pathology on imaging but show specific osseous changes including increased chondrolabral retroversion. This article reviews the relevant anatomy in brief and describes the MRI findings in each type, with the imaging features of the common abnormalities. PMID:21799591

Mode-locking via dissipative Faraday instability

PubMed Central

Tarasov, Nikita; Perego, Auro M.; Churkin, Dmitry V.; Staliunas, Kestutis; Turitsyn, Sergei K.

2016-01-01

Emergence of coherent structures and patterns at the nonlinear stage of modulation instability of a uniform state is an inherent feature of many biological, physical and engineering systems. There are several well-studied classical modulation instabilities, such as Benjamin–Feir, Turing and Faraday instability, which play a critical role in the self-organization of energy and matter in non-equilibrium physical, chemical and biological systems. Here we experimentally demonstrate the dissipative Faraday instability induced by spatially periodic zig-zag modulation of a dissipative parameter of the system—spectrally dependent losses—achieving generation of temporal patterns and high-harmonic mode-locking in a fibre laser. We demonstrate features of this instability that distinguish it from both the Benjamin–Feir and the purely dispersive Faraday instability. Our results open the possibilities for new designs of mode-locked lasers and can be extended to other fields of physics and engineering. PMID:27503708

Mode-locking via dissipative Faraday instability.

PubMed

Tarasov, Nikita; Perego, Auro M; Churkin, Dmitry V; Staliunas, Kestutis; Turitsyn, Sergei K

2016-08-09

Emergence of coherent structures and patterns at the nonlinear stage of modulation instability of a uniform state is an inherent feature of many biological, physical and engineering systems. There are several well-studied classical modulation instabilities, such as Benjamin-Feir, Turing and Faraday instability, which play a critical role in the self-organization of energy and matter in non-equilibrium physical, chemical and biological systems. Here we experimentally demonstrate the dissipative Faraday instability induced by spatially periodic zig-zag modulation of a dissipative parameter of the system-spectrally dependent losses-achieving generation of temporal patterns and high-harmonic mode-locking in a fibre laser. We demonstrate features of this instability that distinguish it from both the Benjamin-Feir and the purely dispersive Faraday instability. Our results open the possibilities for new designs of mode-locked lasers and can be extended to other fields of physics and engineering.

Acute inactivation of the replicative helicase in human cells triggers MCM8–9-dependent DNA synthesis

PubMed Central

Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy; Bhowmick, Rahul; Hickson, Ian D.; Kanemaki, Masato T.

2017-01-01

DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8–9 complex, a paralog of the MCM2–7 replicative helicase. We show that MCM8–9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8–9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8–9 as an alternative replicative helicase. PMID:28487407

Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast

PubMed Central

Ho, Chu Kwen; Mazón, Gerard; Lam, Alicia F.; Symington, Lorraine S.

2010-01-01

Holliday junction (HJ) resolution is required for segregation of chromosomes and for formation of crossovers during homologous recombination. The identity of the resolvase(s) that functions in vivo has yet to be established, although several proteins able to cut HJs in vitro have been identified as candidates in yeasts and mammals. Using an assay to detect unselected products of mitotic recombination we found a significant decrease in crossovers in the Saccharomyces cerevisiae mus81Δ mutant. Yen1 serves a back-up function responsible for resolving intermediates in mus81Δ mutants, or when conversion tracts are short. In the absence of both Mus81 and Yen1 intermediates are not channeled exclusively to non-crossover recombinants, but instead are processed by Pol32-dependent break-induced replication (BIR). The channeling of recombination from reciprocal exchange to BIR results in greatly increased spontaneous loss of heterozygosity (LOH) and chromosome mis-segregation in the mus81Δ yen1Δ mutant, typical of the genomic instability found in tumor cells. PMID:21172663

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors

PubMed Central

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-01-01

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy. DOI: http://dx.doi.org/10.7554/eLife.14709.001 PMID:27304073

A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

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

Ohsaki, Eriko; Ueda, Keiji, E-mail: kueda@virus.me

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components ofmore » the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.« less

The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

PubMed Central

Balajee, Adayabalam S.; Machwe, Amrita; May, Alfred; Gray, Matthew D.; Oshima, Junko; Martin, George M.; Nehlin, Jan O.; Brosh, Robert; Orren, David K.; Bohr, Vilhelm A.

1999-01-01

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype. PMID:10436020

ssDNA damage dependence from singlet oxygen concentration at photodynamic interaction

NASA Astrophysics Data System (ADS)

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

2017-11-01

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

CEP152 is a genome maintenance protein disrupted in Seckel syndrome

PubMed Central

Kalay, Ersan; Yigit, Gökhan; Aslan, Yakup; Brown, Karen E; Pohl, Esther; Bicknell, Louise S; Kayserili, Hülya; Li, Yun; Tüysüz, Beyhan; Nürnberg, Gudrun; Kiess, Wieland; Koegl, Manfred; Baessmann, Ingelore; Buruk, Kurtulus; Toraman, Bayram; Kayipmaz, Saadettin; Kul, Sibel; Ikbal, Mevlit; Turner, Daniel J; Taylor, Martin S; Aerts, Jan; Scott, Carol; Milstein, Karen; Dollfus, Helene; Wieczorek, Dagmar; Brunner, Han G; Hurles, Matthew; Jackson, Andrew P; Rauch, Anita; Nürnberg, Peter; Karagüzel, Ahmet; Wollnik, Bernd

2012-01-01

Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation. PMID:21131973

Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells

PubMed Central

Jia, Pingping; Chastain, Megan; Zou, Ying; Her, Chengtao

2017-01-01

Abstract Aberrant formation of interstitial telomeric sequences (ITSs) promotes genome instabilities. However, it is unclear how aberrant ITS formation is suppressed in human cells. Here, we report that MLH1, a key protein involved in mismatch repair (MMR), suppresses telomeric sequence insertion (TSI) at intra-chromosomal regions. The frequency of TSI can be elevated by double-strand break (DSB) inducer and abolished by ATM/ATR inhibition. Suppression of TSI requires MLH1 recruitment to DSBs, indicating that MLH1's role in DSB response/repair is important for suppressing TSI. Moreover, TSI requires telomerase activity but is independent of the functional status of p53 and Rb. Lastly, we show that TSI is associated with chromosome instabilities including chromosome loss, micronuclei formation and chromosome breakage that are further elevated by replication stress. Our studies uncover a novel link between MLH1, telomerase, telomere and genome stability. PMID:28180301

Human RTEL1 deficiency causes Hoyeraal-Hreidarsson syndrome with short telomeres and genome instability.

PubMed

Le Guen, Tangui; Jullien, Laurent; Touzot, Fabien; Schertzer, Michael; Gaillard, Laetitia; Perderiset, Mylène; Carpentier, Wassila; Nitschke, Patrick; Picard, Capucine; Couillault, Gérard; Soulier, Jean; Fischer, Alain; Callebaut, Isabelle; Jabado, Nada; Londono-Vallejo, Arturo; de Villartay, Jean-Pierre; Revy, Patrick

2013-08-15

Hoyeraal-Hreidarsson syndrome (HHS), a severe variant of dyskeratosis congenita (DC), is characterized by early onset bone marrow failure, immunodeficiency and developmental defects. Several factors involved in telomere length maintenance and/or protection are defective in HHS/DC, underlining the relationship between telomere dysfunction and these diseases. By combining whole-genome linkage analysis and exome sequencing, we identified compound heterozygous RTEL1 (regulator of telomere elongation helicase 1) mutations in three patients with HHS from two unrelated families. RTEL1 is a DNA helicase that participates in DNA replication, DNA repair and telomere integrity. We show that, in addition to short telomeres, RTEL1-deficient cells from patients exhibit hallmarks of genome instability, including spontaneous DNA damage, anaphase bridges and telomeric aberrations. Collectively, these results identify RTEL1 as a novel HHS-causing gene and highlight its role as a genomic caretaker in humans.

Regulated expression of the lncRNA TERRA and its impact on telomere biology.

PubMed

Oliva-Rico, Diego; Herrera, Luis A

2017-10-01

The telomere protects against genomic instability by minimizing the accelerated end resection of the genetic material, a phenomenon that results in severe chromosome instability that could favor the transformation of a cell by enabling the emergence of tumor-promoting mutations. Some mechanisms that avoid this fate, such as capping and loop formation, have been very well characterized; however, telomeric non-coding transcripts, such as long non-coding RNAs (lncRNAs), should also be considered in this context because they play roles in the organization of telomere dynamics, involving processes such as replication, degradation, extension, and heterochromatin stabilization. Although the mechanism through which the expression of telomeric transcripts regulates telomere dynamics is not yet clear, a non-coding RNA component opens the research options in telomere biology and the impact that it can have on telomere-associated diseases such as cancer. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Variation, "evolution", immortality and genetic instabilities in tumour cells.

PubMed

Bignold, L P

2007-08-18

The pathological characteristics of tumour cells often include variation of their histopathological features (i.e. "degrees of de-differentiation") between cases of the same tumour type and between different foci within individual tumours. Usually, only a few cell lines from tumours are immortal. Currently, somatic mutation, replicative infidelity of DNA and aneuploidy are suggested as alternative mechanisms of genomic disturbance underlying tumours. Nevertheless, apart from Hansemann's ideas of "anaplasia" and "de-differentiation" (proposed in the 1890s), and supposed "evolutionary themes" in cancer cell biology, little has been published concerning how histopathologic variation and immortality in tumour cells might arise. This paper reviews applications of the concepts of "variation" to tumours, including concepts of "evolution" and "cellular Darwinism". It is proposed that combinations of somatic mutation, DNA replicative infidelity and aneuploidy may explain the variabilities in tumours, and provide immortality in occasional tumour cells. A possible model involves (i) an initial somatic mutation causing reduced replicative fidelity of DNA, which could be variable in intensity, and thus give rise to variations between cases; (ii) a phase of replicative infidelity of DNA causing daughter cells lines to develop various abnormalities to different degrees, and hence provide for variation between areas of the same tumour. As a last event (iii) occasional asymmetric chromosomal distributions (aneuploidy) might "refresh" the ability of a daughter cell to replicate DNA faithfully causing them to become immortal. Thus extensively mutant and variable, hyperploid, and occasionally immortal cells might arise.

Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

PubMed Central

Hayes, Sidney; Erker, Craig; Horbay, Monique A.; Marciniuk, Kristen; Wang, Wen; Hayes, Connie

2013-01-01

The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step. PMID:23389467

Variety of DNA Replication Activity Among Cyanobacteria Correlates with Distinct Respiration Activity in the Dark.

PubMed

Ohbayashi, Ryudo; Yamamoto, Jun-Ya; Watanabe, Satoru; Kanesaki, Yu; Chibazakura, Taku; Miyagishima, Shin-Ya; Yoshikawa, Hirofumi

2017-02-01

Cyanobacteria exhibit light-dependent cell growth since most of their cellular energy is obtained by photosynthesis. In Synechococcus elongatus PCC 7942, one of the model cyanobacteria, DNA replication depends on photosynthetic electron transport. However, the critical signal for the regulatory mechanism of DNA replication has not been identified. In addition, conservation of this regulatory mechanism has not been investigated among cyanobacteria. To understand this regulatory signal and its dependence on light, we examined the regulation of DNA replication under both light and dark conditions among three model cyanobacteria, S. elongatus PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. Interestingly, DNA replication activity in Synechocystis and Anabaena was retained when cells were transferred to the dark, although it was drastically decreased in S. elongatus. Glycogen metabolism and respiration were higher in Synechocystis and Anabaena than in S. elongatus in the dark. Moreover, DNA replication activity in Synechocystis and Anabaena was reduced to the same level as that in S. elongatus by inhibition of respiratory electron transport after transfer to the dark. These results demonstrate that there is disparity in DNA replication occurring in the dark among cyanobacteria, which is caused by the difference in activity of respiratory electron transport. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Stabilization of hydrodynamic flows by small viscosity variations.

PubMed

Govindarajan, Rama; L'vov, Victor S; Procaccia, Itamar; Sameen, A

2003-02-01

Motivated by the large effect of turbulent drag reduction by minute concentrations of polymers, we study the effects of a weakly space-dependent viscosity on the stability of hydrodynamic flows. In a recent paper [Phys. Rev. Lett. 87, 174501, (2001)], we exposed the crucial role played by a localized region where the energy of fluctuations is produced by interactions with the mean flow (the "critical layer"). We showed that a layer of a weakly space-dependent viscosity placed near the critical layer can have a very large stabilizing effect on hydrodynamic fluctuations, retarding significantly the onset of turbulence. In this paper we extend these observations in two directions: first we show that the strong stabilization of the primary instability is also obtained when the viscosity profile is realistic (inferred from simulations of turbulent flows with a small concentration of polymers). Second, we analyze the secondary instability (around the time-dependent primary instability) and find similar strong stabilization. Since the secondary instability develops around a time-dependent solution and is three dimensional, this brings us closer to the turbulent case. We reiterate that the large effect is not due to a modified dissipation (as is assumed in some theories of drag reduction), but due to reduced energy intake from the mean flow to the fluctuations. We propose that similar physics act in turbulent drag reduction.

Radiation-induced genomic instability

NASA Technical Reports Server (NTRS)

Kronenberg, A.

1994-01-01

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

Transverse Mode Coupling Instability of the Bunch with Oscillating Wake Field and Space Charge

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

Balbekov, V.

Transverse mode coupling instability of a single bunch caused by oscillating wake field is considered in the paper. The instability threshold is found at different frequencies of the wake with space charge tune shift taken into account. The wake phase advance in the bunch length from 0 up tomore » $$4\\pi$$ is investigated. It is shown that the space charge can push the instability threshold up or down dependent on the phase advance. Transition region is investigated thoroughly, and simple asymptotic formulas for the threshold are represented.« less

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

PubMed

Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses. Copyright © 2017 American Society for Microbiology.

The Influence of Hydroxylation on Maintaining CpG Methylation Patterns: A Hidden Markov Model Approach.

PubMed

Giehr, Pascal; Kyriakopoulos, Charalampos; Ficz, Gabriella; Wolf, Verena; Walter, Jörn

2016-05-01

DNA methylation and demethylation are opposing processes that when in balance create stable patterns of epigenetic memory. The control of DNA methylation pattern formation by replication dependent and independent demethylation processes has been suggested to be influenced by Tet mediated oxidation of 5mC. Several alternative mechanisms have been proposed suggesting that 5hmC influences either replication dependent maintenance of DNA methylation or replication independent processes of active demethylation. Using high resolution hairpin oxidative bisulfite sequencing data, we precisely determine the amount of 5mC and 5hmC and model the contribution of 5hmC to processes of demethylation in mouse ESCs. We develop an extended hidden Markov model capable of accurately describing the regional contribution of 5hmC to demethylation dynamics. Our analysis shows that 5hmC has a strong impact on replication dependent demethylation, mainly by impairing methylation maintenance.

Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner.

PubMed

Krejciova, Zuzana; Alibhai, James; Zhao, Chen; Krencik, Robert; Rzechorzek, Nina M; Ullian, Erik M; Manson, Jean; Ironside, James W; Head, Mark W; Chandran, Siddharthan

2017-12-04

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype-dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery. © 2017 Krejciova et al.

RAD51 and RTEL1 compensate telomere loss in the absence of telomerase.

PubMed

Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon; White, Charles I; Gallego, Maria E

2018-03-16

Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.

Optimal parameters of leader development in lightning

NASA Technical Reports Server (NTRS)

Petrov, N. I.; Petrova, G. N.

1991-01-01

The dependences between the different parameters of a leader in lightning are obtained theoretically. The physical mechanism of the instability leading to the formation of the streamer zone is proposed. The instability has the wave nature and is caused by the self-influence effects of the space charge. Using a stability condition of the leader propagation, a dependence is obtained between the current across the leader head and its velocity of motion. The dependence of the streamer zone length on the gap length is also obtained. It is shown that the streamer zone length is saturated with the increasing of the gap length. A comparison between the obtained dependences and the experimental data is presented.

Radiation-induced genomic instability: radiation quality and dose response

NASA Technical Reports Server (NTRS)

Smith, Leslie E.; Nagar, Shruti; Kim, Grace J.; Morgan, William F.

2003-01-01

Genomic instability is a term used to describe a phenomenon that results in the accumulation of multiple changes required to convert a stable genome of a normal cell to an unstable genome characteristic of a tumor. There has been considerable recent debate concerning the importance of genomic instability in human cancer and its temporal occurrence in the carcinogenic process. Radiation is capable of inducing genomic instability in mammalian cells and instability is thought to be the driving force responsible for radiation carcinogenesis. Genomic instability is characterized by a large collection of diverse endpoints that include large-scale chromosomal rearrangements and aberrations, amplification of genetic material, aneuploidy, micronucleus formation, microsatellite instability, and gene mutation. The capacity of radiation to induce genomic instability depends to a large extent on radiation quality or linear energy transfer (LET) and dose. There appears to be a low dose threshold effect with low LET, beyond which no additional genomic instability is induced. Low doses of both high and low LET radiation are capable of inducing this phenomenon. This report reviews data concerning dose rate effects of high and low LET radiation and their capacity to induce genomic instability assayed by chromosomal aberrations, delayed lethal mutations, micronuclei and apoptosis.

Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

PubMed Central

Meier, Jeffery L.; Keller, Michael J.; McCoy, James J.

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication. PMID:11739696

Role of MAPK/MNK1 signaling in virus replication.

PubMed

Kumar, Ram; Khandelwal, Nitin; Thachamvally, Riyesh; Tripathi, Bhupendra Nath; Barua, Sanjay; Kashyap, Sudhir Kumar; Maherchandani, Sunil; Kumar, Naveen

2018-06-01

Viruses are obligate intracellular parasites; they heavily depend on the host cell machinery to effectively replicate and produce new progeny virus particles. Following viral infection, diverse cell signaling pathways are initiated by the cells, with the major goal of establishing an antiviral state. However, viruses have been shown to exploit cellular signaling pathways for their own effective replication. Genome-wide siRNA screens have also identified numerous host factors that either support (proviral) or inhibit (antiviral) virus replication. Some of the host factors might be dispensable for the host but may be critical for virus replication; therefore such cellular factors may serve as targets for development of antiviral therapeutics. Mitogen activated protein kinase (MAPK) is a major cell signaling pathway that is known to be activated by diverse group of viruses. MAPK interacting kinase 1 (MNK1) has been shown to regulate both cap-dependent and internal ribosomal entry sites (IRES)-mediated mRNA translation. In this review we have discuss the role of MAPK in virus replication, particularly the role of MNK1 in replication and translation of viral genome. Copyright © 2018 Elsevier B.V. All rights reserved.

GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

PubMed

Santillan, Beatriz A; Moye, Christopher; Mittelman, David; Wilson, John H

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

PubMed Central

Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

Emotionally Up and Down, Behaviorally To and Fro: Drinking Motives Mediate the Synergistic Effects of Urgency and Emotional Instability on Alcohol Outcomes.

PubMed

Dvorak, Robert D; Kuvaas, Nicholas J; Lamis, Dorian A; Pearson, Matthew R; Stevenson, Brittany L

2015-01-01

Emotional and behavioral regulation has been linked to coping and enhancement motives and associated with different patterns of alcohol use and problems. The current studies examined emotional instability, urgency, and internal drinking motives as predictors of alcohol dependence symptoms as well as the likelihood and severity of Diagnostic and Statistical Manual of Mental Disorders-5th editionAlcohol Use Disorder (AUD). In Study 1, college drinkers (n = 621) completed alcohol involvement and behavioral/emotional functioning assessments. There was an indirect association between emotional instability and dependence symptoms via both coping and enhancement drinking motives which was potentiated by trait urgency. In Study 2, college drinkers (n = 510) completed alcohol involvement, behavioral/emotional functioning, and AUD criteria assessments. A significant indirect effect from emotional instability to the likelihood of meeting AUD criteria, via drinking to cope was found, again potentiated by urgency. © The Author(s) 2016.

Antiviral effects of artesunate on polyomavirus BK replication in primary human kidney cells.

PubMed

Sharma, Biswa Nath; Marschall, Manfred; Henriksen, Stian; Rinaldo, Christine Hanssen

2014-01-01

Polyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 μM. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentration-dependent manner. Artesunate at 10 μM reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 μM reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2'-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G0 or G2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 μM were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions.

Antiviral Effects of Artesunate on Polyomavirus BK Replication in Primary Human Kidney Cells

PubMed Central

Sharma, Biswa Nath; Marschall, Manfred; Henriksen, Stian

2014-01-01

Polyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 μM. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentration-dependent manner. Artesunate at 10 μM reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 μM reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2′-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G0 or G2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 μM were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions. PMID:24145549

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

PubMed Central

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

Filamentation instability of a fast electron beam in a dielectric target.

PubMed

Debayle, A; Tikhonchuk, V T

2008-12-01

High-intensity laser-matter interaction is an efficient method for high-current relativistic electron beam production. At current densities exceeding a several kA microm{-2} , the beam propagation is maintained by an almost complete current neutralization by the target electrons. In such a geometry of two oppositely directed flows, beam instabilities can develop, depending on the target and the beam parameters. The present paper proposes an analytical description of the filamentation instability of an electron beam propagating through an insulator target. It is shown that the collisionless and resistive instabilities enter into competition with the ionization instability. This latter process is dominant in insulator targets where the field ionization by the fast beam provides free electrons for the neutralization current.

Multiple frequency radar observations of high-latitude E region irregularities in the HF modified ionosphere

NASA Technical Reports Server (NTRS)

Noble, S. T.; Gordon, W. E.; Djuth, F. T.; Jost, R. J.; Hedberg, A.

1987-01-01

This paper discusses the results of the September 1983 observations of artificial field-aligned irregularities (AFAIs) in the Tromso, Norway region, made by backscatter radars operating at 46.9, 143.8, 21.4, and 140.0 MHz. Four classes of resonant instability processes at work in the E and F regions are examined in detail: (1) the coupling of parametric decay instability waves across geomagnetic field lines, (2) thermal parametric instability, (3) four-wave interaction thermal parametric instability, and (4) the resonance instability. The characteristics of the AFAI scatter are described, with special attention given to the growth and decay time constants, functional dependence on the heater power and polarization, and the scattering cross sections of the irregularities.

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

Gruenwald, J., E-mail: johannes.gruenwald@inp-greifswald.de; Fröhlich, M.

A model of the behavior of transit time instabilities in an electrostatic confinement fusion reactor is presented in this letter. It is demonstrated that different modes are excited within the spherical cathode of a Farnsworth fusor. Each of these modes is dependent on the fusion products as well as the acceleration voltage applied between the two electrodes and they couple to a resulting oscillation showing non-linear beat phenomena. This type of instability is similar to the transit time instability of electrons between two resonant surfaces but the presence of ions and the occurring fusion reactions alter the physics of thismore » instability considerably. The physics of this plasma instability is examined in detail for typical physical parameter ranges of electrostatic confinement fusion devices.« less

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

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

Malkinson, Mertyn; Winocour, Ernest

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM.more » AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.« less

Esophageal squamous cell carcinomas with DNA replication errors (RER+) are associated with p16/pRb loss and wild-type p53.

PubMed

Mathew, R; Arora, S; Mathur, M; Chattopadhyay, T K; Ralhan, R

2001-10-01

Microsatellite instability (MSI) as a determinant of propensity to esophageal squamous cell carcinoma (ESCC) at seven microsatellite markers at 2p (2p15-16), 3p (3p13, 3p14.1-3, 3p25, and 3p26) and 16q (16q12.1-3) was investigated to analyze their putative role as indicators of predisposition to esophageal malignancies. Seven microsatellite loci were amplified by polymerase chain reaction, from surgically resected tumor tissues from 30 ESCC patients from Indian population, to assess the loss of heterozygosity (LOH) and replication error repeats (RER) and to correlate these alterations with aberrations in major cell cycle regulatory proteins and histopathological parameters. LOH and RER analyses at these loci demonstrated moderate microsatellite alterations, suggesting the involvement of MSI in esophageal tumorigenesis in a subset of the Indian population. MSI, defined as RER in at least two or more of the loci studied, was observed in ten of 30 (33%) patients. Twenty-two of 30 patients (73%) showed LOH at one or more loci, while 17 of the 30 patients (60%) showed RER in at least one of the loci studied. RER-positive patients showed a trend towards better prognosis when compared to RER-negative patients. MSI demonstrated a significant association with concomitant loss of p16 and pRb (p16-/pRb- phenotype) (P=0.046). Interestingly, we observed an inverse correlation between MSI and p53 mutations (P=0.03) suggesting that MSI may provide a p53-independent pathway for esophageal tumorigenesis in RER+ patients. MSI showed a trend towards longer survival and absence of distant organ metastasis (P=0.06). The present study demonstrates the probable role of MSI in esophageal squamous cell carcinoma in the Indian population. Instability associated with the repetitive sequences--the revealing marks of loss of DNA replication fidelity may serve as an indicator of predisposition to esophageal cancer.

R-loops: targets for nuclease cleavage and repeat instability.

PubMed

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner.

PubMed

Wiedemann, Eva-Maria; Peycheva, Mihaela; Pavri, Rushad

2016-12-13

Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2017-03-01

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

Selfish cells in altruistic cell society - a theoretical oncology.

PubMed

Chigira, M

1993-09-01

In multicellular organisms, internal evolution of individual cells is strictly forbidden and 'evolutional' DNA replication should be performed only by the sexual reproduction system. Wholistic negative control system called 'homeostasis' serves all service to germ line cells. All somatic cells are altruistic to the germ line cells. However, in malignant tumors, it seems that individual cells replicate and behave 'selfishly' and evolve against the internal microenvironment. Tumor cells only express the occult selfishness which is programmed in normal cells a priori. This phenomenon is based on the failure of identical DNA replication, and results in 'autonomy' and 'anomie' of cellular society as shown in tumor cells. Genetic programs of normal cells connote this cellular autonomy and anomie introduced by the deletion of regulators on structure genes. It is rather paradoxical that the somatic cells get their freedom from wholistic negative regulation programmed internally. However, this is not a true paradox, since multicellular organisms have clearly been evolved from 'monads' in which cells proliferate without wholistic regulation. Somatic cells revolt against germ cell DNA, called 'selfish replicator' by Dawkins. It is an inevitable destiny that the 'selfishness' coded in genome should be revenged by itself. Selfish replicator in germ cell line should be revolted by its selfishness in the expansion of somatic cells, since they have an orthogenesis to get more selfishness in order to increase their genome. Tumor heterogeneity and progression can be fully explained by this self-contradictory process which produces heterogeneous gene copies different from the original clone in the tumor, although 'selfish' gene replication is the final target of being. Furthermore, we have to discard the concept of clonality of tumor cells since genetic instability is a fundamental feature of tumors. Finally, tumor cells and proto-oncogenes can be considered as the ultimate parasite to germ line cells.

A replication of the relationship between elderly suicides rates and elderly dependency ratios: a cross-national study.

PubMed

Shah, Ajit

2010-01-01

A positive correlation between elderly dependency ratios and elderly suicide rates has been observed using one-year cross-sectional data on elderly suicide rates. A cross-national study designed to replicate this positive correlation between elderly dependency ratios and elderly suicide rates was undertaken by: (i) using one-year average of five years data on suicide rates; and (ii) using more recent data on both elderly suicide rates and elderly dependency ratios. Data on elderly suicide rates, and the total number of elderly and young people was ascertained from the World Health Organization website. The main findings were of significant positive correlations between elderly dependency ratios and suicide rates in both sexes in both the elderly age-bands (65-74 years and 75+ years). The replication of the positive correlations between elderly dependency ratios and elderly suicide rates by using one-year average of five years data on suicide rates suggests that this relationship is robust and accurate. ‎

A replication of the relationship between elderly suicides rates and elderly dependency ratios: cross-national study

PubMed Central

Shah, Ajit

2010-01-01

Abstract: Background: A positive correlation between elderly dependency ratios and elderly suicide rates has been observed using one-year cross-sectional data on elderly suicide rates. Methods: A cross-national study designed to replicate this positive correlation between elderly dependency ratios and elderly suicide rates was undertaken by: (i) using one-year average of five years data on suicide rates; and (ii) using more recent data on both elderly suicide rates and elderly dependency ratios. Data on elderly suicide rates, and the total number of elderly and young people was ascertained from the World Health Organization website. Results: The main findings were of significant positive correlations between elderly dependency ratios and suicide rates in both sexes in both the elderly age-bands (65-74 years and 75+ years). Conclusions: The replication of the positive correlations between elderly dependency ratios and elderly suicide rates by using one-year average of five years data on suicide rates suggests that this relationship is robust and accurate. PMID:21483194

Electron Debye scale Kelvin-Helmholtz instability: Electrostatic particle-in-cell simulations

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

Lee, Sang-Yun; Lee, Ensang, E-mail: eslee@khu.ac.kr; Kim, Khan-Hyuk

2015-12-15

In this paper, we investigated the electron Debye scale Kelvin-Helmholtz (KH) instability using two-dimensional electrostatic particle-in-cell simulations. We introduced a velocity shear layer with a thickness comparable to the electron Debye length and examined the generation of the KH instability. The KH instability occurs in a similar manner as observed in the KH instabilities in fluid or ion scales producing surface waves and rolled-up vortices. The strength and growth rate of the electron Debye scale KH instability is affected by the structure of the velocity shear layer. The strength depends on the magnitude of the velocity and the growth ratemore » on the velocity gradient of the shear layer. However, the development of the electron Debye scale KH instability is mainly determined by the electric field generated by charge separation. Significant mixing of electrons occurs across the shear layer, and a fraction of electrons can penetrate deeply into the opposite side fairly far from the vortices across the shear layer.« less

Pulse bifurcations and instabilities in an excitable medium: Computations in finite ring domains

NASA Astrophysics Data System (ADS)

Or-Guil, M.; Krishnan, J.; Kevrekidis, I. G.; Bär, M.

2001-10-01

We investigate the instabilities and bifurcations of traveling pulses in a model excitable medium; in particular, we discuss three different scenarios involving either the loss of stability or disappearance of stable pulses. In numerical simulations beyond the instabilities we observe replication of pulses (``backfiring'') resulting in complex periodic or spatiotemporally chaotic dynamics as well as modulated traveling pulses. We approximate the linear stability of traveling pulses through computations in a finite albeit large domain with periodic boundary conditions. The critical eigenmodes at the onset of the instabilities are related to the resulting spatiotemporal dynamics and ``act'' upon the back of the pulses. The first scenario has been analyzed earlier [M. G. Zimmermann et al., Physica D 110, 92 (1997)] for high excitability (low excitation threshold): it involves the collision of a stable pulse branch with an unstable pulse branch in a so-called T point. In the framework of traveling wave ordinary differential equations, pulses correspond to homoclinic orbits and the T point to a double heteroclinic loop. We investigate this transition for a pulse in a domain with finite length and periodic boundary conditions. Numerical evidence of the proximity of the infinite-domain T point in this setup appears in the form of two saddle node bifurcations. Alternatively, for intermediate excitation threshold, an entire cascade of saddle nodes causing a ``spiraling'' of the pulse branch appears near the parameter values corresponding to the infinite-domain T point. Backfiring appears at the first saddle-node bifurcation, which limits the existence region of stable pulses. The third case found in the model for large excitation threshold is an oscillatory instability giving rise to ``breathing,'' traveling pulses that periodically vary in width and speed.

A 4-Week Neuromuscular Training Program and Gait Patterns at the Ankle Joint

PubMed Central

Coughlan, Garrett; Caulfield, Brian

2007-01-01

Context: Previous research into the rehabilitation of ankle sprains has primarily focused on outcome measures that do not replicate functional activities, thus making it difficult to extrapolate the results relative to the weight-bearing conditions under which most ankle sprains occur. Objective: To measure the effects of a training program on gait during walking and running in an active athletic population. Design: Matched-pairs, controlled trial. Setting: University motion analysis laboratory. Patients or Other Participants: Ten subjects from an athletic population (7 healthy, 3 with functional ankle instability: age = 25.8 ± 3.9 years, height = 177.6 ± 6.1 cm, mass = 66.8 ± 7.4 kg) and 10 controls matched for age, sex, activity, and ankle instability (7 healthy, 3 with functional ankle instability: age = 27.4 ± 5.8 years, height = 178.7 ± 10.8 cm, mass = 71.6 ± 10.0 kg). Intervention(s): A 4-week neuromuscular training program undertaken by the treatment group. Main Outcome Measure(s): We measured ankle position and velocity in the frontal (x) and sagittal (y) planes in all subjects during treadmill walking and running for the periods 100 milliseconds before heel strike, at heel strike, and 100 milliseconds after heel strike. Results: A 4-week neuromuscular training program resulted in no significant changes in ankle position or velocity during treadmill walking and running. Conclusions: The mechanisms by which neuromuscular training improves function in normal subjects and those with functional ankle instability do not appear to result in measurable changes in gait kinematics. Our findings raise issues regarding methods of ankle sprain rehabilitation and the measurement of their effectiveness in improving functional activities. Further research in a larger population with functional ankle instability is necessary. PMID:17597944

Kelvin-Helmholtz instability of counter-rotating discs

NASA Astrophysics Data System (ADS)

Quach, Dan; Dyda, Sergei; Lovelace, Richard V. E.

2015-01-01

Observations of galaxies and models of accreting systems point to the occurrence of counter-rotating discs where the inner part of the disc (r < r0) is corotating and the outer part is counter-rotating. This work analyses the linear stability of radially separated co- and counter-rotating thin discs. The strong instability found is the supersonic Kelvin-Helmholtz instability. The growth rates are of the order of or larger than the angular rotation rate at the interface. The instability is absent if there is no vertical dependence of the perturbation. That is, the instability is essentially three dimensional. The non-linear evolution of the instability is predicted to lead to a mixing of the two components, strong heating of the mixed gas, and vertical expansion of the gas, and annihilation of the angular momenta of the two components. As a result, the heated gas will free-fall towards the disc's centre over the surface of the inner disc.

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

Niknam, A. R., E-mail: a-niknam@sbu.ac.ir; Rastbood, E.; Khorashadizadeh, S. M.

The dielectric permittivity tensor of a magnetoactive current-driven plasma is obtained by employing the kinetic theory based on the Vlasov equation and Lorentz transformation formulas with an emphasize on the q-nonextensive statistics. By deriving the q-generalized dispersion relation of the low frequency modes in this plasma system, the possibility and properties of filamentation and ion acoustic instabilities are then studied. It is shown that the occurrence and the growth rate of these instabilities depend strongly on the nonextensive parameters, external magnetic field strength, and drift velocity. It is observed that the growth rate of ion acoustic instability is affected bymore » the magnetic field strength much more than that of the filamentation instability in the low frequency range. The external magnetic field facilitates the development of the ion-acoustic instability. It is also shown that the filamentation is the dominant instability only for the high value of drift velocity.« less

Sedimentation and gravitational instability of Escherichia coli Suspension

NASA Astrophysics Data System (ADS)

Douarche, Carine; Salin, Dominique; Collaboration between Laboratory FAST; LPS Collaboration

2016-11-01

The successive run and tumble of Escherichia coli bacteria provides an active matter suspension of rod-like particles with a large swimming diffusion. As opposed to inactive elongated particles, this diffusion prevents clustering and instability in the gravity field. We measure the time dependent E . coli concentration profile during their sedimentation. After some hours, due to the dioxygen consumption, a motile / non-motile front forms leading to a Rayleigh-Taylor type gravitational instability. Analyzing both sedimentation and instability in the framework of active particle suspensions, we can measure the relevant bacteria hydrodynamic characteristics such as its single particle sedimentation velocity and its hindrance volume.

Studies of fluid instabilities in flows of lava and debris

NASA Technical Reports Server (NTRS)

Fink, Jonathan H.

1987-01-01

At least two instabilities have been identified and utilized in lava flow studies: surface folding and gravity instability. Both lead to the development of regularly spaced structures on the surfaces of lava flows. The geometry of surface folds have been used to estimate the rheology of lava flows on other planets. One investigation's analysis assumed that lava flows have a temperature-dependent Newtonian rheology, and that the lava's viscosity decreased exponentially inward from the upper surface. The author reviews studies by other investigators on the analysis of surface folding, the analysis of Taylor instability in lava flows, and the effect of surface folding on debris flows.

Instability of meridional axial system in f( R) gravity

NASA Astrophysics Data System (ADS)

Sharif, M.; Yousaf, Z.

2015-05-01

We analyze the dynamical instability of a non-static reflection axial stellar structure by taking into account the generalized Euler equation in metric f( R) gravity. Such an equation is obtained by contracting the Bianchi identities of the usual anisotropic and effective stress-energy tensors, which after using a radial perturbation technique gives a modified collapse equation. In the realm of the gravity model, we investigate instability constraints at Newtonian and post-Newtonian approximations. We find that the instability of a meridional axial self-gravitating system depends upon the static profile of the structure coefficients, while f( R) extra curvature terms induce the stability of the evolving celestial body.

Onset of dissolution-driven instabilities in fluids with nonmonotonic density profile

NASA Astrophysics Data System (ADS)

Jafari Raad, Seyed Mostafa; Hassanzadeh, Hassan

2015-11-01

Analog systems have recently been used in several experiments in the context of convective mixing of C O2 . We generalize the nonmonotonic density dependence of the growth of instabilities and provide a scaling relation for the onset of instability. The results of linear stability analysis and direct numerical simulations show that these fluids do not resemble the dynamics of C O2 -water convective instabilities. A typical analog system, such as water-propylene glycol, is found to be less unstable than C O2 -water. These results provide a basis for further research and proper selection of analog systems and are essential to the interpretation of experiments.

Competing growth processes induced by next-nearest-neighbor interactions: Effects on meandering wavelength and stiffness

NASA Astrophysics Data System (ADS)

Blel, Sonia; Hamouda, Ajmi BH.; Mahjoub, B.; Einstein, T. L.

2017-02-01

In this paper we explore the meandering instability of vicinal steps with a kinetic Monte Carlo simulations (kMC) model including the attractive next-nearest-neighbor (NNN) interactions. kMC simulations show that increase of the NNN interaction strength leads to considerable reduction of the meandering wavelength and to weaker dependence of the wavelength on the deposition rate F. The dependences of the meandering wavelength on the temperature and the deposition rate obtained with simulations are in good quantitative agreement with the experimental result on the meandering instability of Cu(0 2 24) [T. Maroutian et al., Phys. Rev. B 64, 165401 (2001), 10.1103/PhysRevB.64.165401]. The effective step stiffness is found to depend not only on the strength of NNN interactions and the Ehrlich-Schwoebel barrier, but also on F. We argue that attractive NNN interactions intensify the incorporation of adatoms at step edges and enhance step roughening. Competition between NNN and nearest-neighbor interactions results in an alternative form of meandering instability which we call "roughening-limited" growth, rather than attachment-detachment-limited growth that governs the Bales-Zangwill instability. The computed effective wavelength and the effective stiffness behave as λeff˜F-q and β˜eff˜F-p , respectively, with q ≈p /2 .

Delay-dependent stability and added damping of SDOF real-time dynamic hybrid testing

NASA Astrophysics Data System (ADS)

Chi, Fudong; Wang, Jinting; Jin, Feng

2010-09-01

It is well-recognized that a transfer system response delay that reduces the test stability inevitably exists in real-time dynamic hybrid testing (RTDHT). This paper focuses on the delay-dependent stability and added damping of SDOF systems in RTDHT. The exponential delay term is transferred into a rational fraction by the Padé approximation, and the delay-dependent stability conditions and instability mechanism of SDOF RTDHT systems are investigated by the root locus technique. First, the stability conditions are discussed separately for the cases of stiffness, mass, and damping experimental substructure. The use of root locus plots shows that the added damping effect and instability mechanism for mass are different from those for stiffness. For the stiffness experimental substructure case, the instability results from the inherent mode because of an obvious negative damping effect of the delay. For the mass case, the delay introduces an equivalent positive damping into the inherent mode, and instability occurs at an added high frequency mode. Then, the compound stability condition is investigated for a general case and the results show that the mass ratio may have both upper and lower limits to remain stable. Finally, a high-emulational virtual shaking table model is built to validate the stability conclusions.

Review of Inertial Confinement Fusion

NASA Astrophysics Data System (ADS)

Haines, M. G.

The physics of inertial confinement fusion is reviewed. The trend to short-wavelength lasers is argued, and the distinction between direct and indirect (soft X-ray) drive is made. Key present issues include the non-linear growth of Rayleigh-Taylor (R-T) instabilities, the seeding of this instability by the initial laser imprint, the relevance of self-generated magnetic fields, and the importance of parametric instabilities (stimulated Brillouin and Raman scattering) in gas-filled hohlraums. Experiments are reviewed which explore the R-T instability in both planar and converging geometry. The employment of various optical smoothing techniques is contrasted with the overcoating of the capsule by gold coated plastic foams to reduce considerably the imprint problem. The role of spontaneously generated magnetic fields in non-symmetric plasmas is discussed. Recent hohlraum compression results are presented together with gas bag targets which replicate the long-scale-length low density plasmas expected in NIF gas filled hohlraums. The onset of first Brillouin and then Raman scattering is observed. The fast ignitor scheme is a proposal to use an intense short pulse laser to drill a hole through the coronal plasma and then, with laser excited fast electrons, create a propagating thermonuclear spark in a dense, relatively cold laser-compressed target. Some preliminary results of laser hole drilling and 2-D and 3-D PIC simulations of this and the > 10^8 Gauss self-generated magnetic fields are presented. The proposed National Ignition Facility (NIF) is described.

Genome-wide analysis of signal transducers and regulators of mitochondrial dysfunction in Saccharomyces cerevisiae.

PubMed

Singh, Keshav K; Rasmussen, Anne Karin; Rasmussen, Lene Juel

2004-04-01

Mitochondrial dysfunction is a hallmark of cancer cells. However, genetic response to mitochondrial dysfunction during carcinogenesis is unknown. To elucidate genetic response to mitochondrial dysfunction we used Saccharomyces cerevisiae as a model system. We analyzed genome-wide expression of nuclear genes involved in signal transduction and transcriptional regulation in a wild-type yeast and a yeast strain lacking the mitochondrial genome (rho(0)). Our analysis revealed that the gene encoding cAMP-dependent protein kinase subunit 3 (PKA3) was upregulated. However, the gene encoding cAMP-dependent protein kinase subunit 2 (PKA2) and the VTC1, PTK2, TFS1, CMK1, and CMK2 genes, involved in signal transduction, were downregulated. Among the known transcriptional factors, OPI1, MIG2, INO2, and ROX1 belonged to the upregulated genes, whereas MSN4, MBR1, ZMS1, ZAP1, TFC3, GAT1, ADR1, CAT8, and YAP4 including RFA1 were downregulated. RFA1 regulates DNA repair genes at the transcriptional level. RFA is also involved directly in DNA recombination, DNA replication, and DNA base excision repair. Downregulation of RFA1 in rho(0) cells is consistent with our finding that mitochondrial dysfunction leads to instability of the nuclear genome. Together, our data suggest that gene(s) involved in mitochondria-to-nucleus communication play a role in mutagenesis and may be implicated in carcinogenesis.

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

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

Endsley, Mark A., E-mail: maendsle@utmb.edu; Somasunderam, Anoma D., E-mail: asomasun@utmb.edu; Li, Guangyu, E-mail: LIG001@mail.etsu.edu

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizesmore » with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.« less

Interpersonal stressors and negative affect in individuals with borderline personality disorder and community adults in daily life: A replication and extension.

PubMed

Hepp, Johanna; Lane, Sean P; Wycoff, Andrea M; Carpenter, Ryan W; Trull, Timothy J

2018-02-01

Affective instability and interpersonal stress are key features of borderline personality disorder (BPD). They were shown to covary in the daily lives of patients in a recent ambulatory assessment study (Hepp et al., 2017) that observed comparatively larger positive associations between interpersonal stressors and negative affect in individuals with BPD than those with depressive disorders. The present study sought to replicate these findings, collecting data on hostility, sadness, fear, and rejection or disagreement events from 56 BPD and 60 community control participants for 21 days, 6 times a day. Using identical statistical procedures, the positive associations between momentary rejection/disagreement and hostility, sadness, and fear were replicated. Again replicating the original study, the rejection-hostility, rejection-sadness, and disagreement-hostility associations were significantly stronger in the BPD group. Time-lagged analyses extended the original study, revealing that rejection was associated with subsequent hostility and sadness more strongly in the BPD group, as was disagreement with subsequent hostility and fear. Though small, we argue that the observed group differences reflect meaningful pervasive responses in a daily life context. Future research should consider these when implementing affect regulation strategies that are applicable in interpersonal contexts for all individuals, but particularly those with BPD. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

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

PubMed Central

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

2016-01-01

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

Hydrodynamic Model for Density Gradients Instability in Hall Plasmas Thrusters

NASA Astrophysics Data System (ADS)

Singh, Sukhmander

2017-10-01

There is an increasing interest for a correct understanding of purely growing electromagnetic and electrostatic instabilities driven by a plasma gradient in a Hall thruster devices. In Hall thrusters, which are typically operated with xenon, the thrust is provided by the acceleration of ions in the plasma generated in a discharge chamber. The goal of this paper is to study the instabilities due to gradients of plasma density and conditions for the growth rate and real part of the frequency for Hall thruster plasmas. Inhomogeneous plasmas prone a wide class of eigen modes induced by inhomogeneities of plasma density and called drift waves and instabilities. The growth rate of the instability has a dependences on the magnetic field, plasma density, ion temperature and wave numbers and initial drift velocities of the plasma species.

Dynamic Stabilization of the Ablative Rayleigh-Taylor Instability for Heavy Ion Fusion

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

Qin, Hong; Davidson, Ronald C.; Logan, B. Grant

2012-10-04

Dynamic stabilization of the ablative Rayleigh-Taylor instability of a heavy ion fusion target induced by a beam wobbling system is studied. Using a sharp-boundary model and Courant-Synder theory, it is shown, with an appropriately chosen modulation waveform, that the instability can be sta- bilized in certain parameter regimes. It is found that the stabilization e ect has a strong dependence on the modulation frequency and the waveform. Modulation with frequency comparable to the instability growth rate is the most e ective in terms of stabilizing the instability. A modulation with two frequency components can result in a reduction of themore » growth rate larger than the sum of that due to the two components when applied separately.« less

Interface instabilities during displacements of two miscible fluids in a vertical pipe

NASA Astrophysics Data System (ADS)

Scoffoni, J.; Lajeunesse, E.; Homsy, G. M.

2001-03-01

We study experimentally the downward vertical displacement of one miscible fluid by another in a vertical pipe at sufficiently high velocities for diffusive effects to be negligible. For certain viscosity ratios and flow rates, the interface between the two fluids can destabilize. We determine the dimensionless flow rate Uc above which the instability is triggered and its dependence on the viscous ratio M, resulting in a stability map Uc=Uc(M). Two different instability modes have been observed: an asymmetric "corkscrew" mode and an axisymmetric one. We remark that the latter is always eventually disturbed by "corkscrew" type instabilities. We speculate that these instabilities are driven by the viscosity stratification and are analogous to those already observed in core annular flows of immiscible fluids.

The Regulatory Interactions of p21 and PCNA in Human Breast Cancer

DTIC Science & Technology

2002-07-01

Proliferating cell nuclear antigen (PCNA) is a multifunctional enzyme involved in multiple cellular processes including DNA replication and repair...During DNA replication , PCNA function as an accessory factor- for the DNA polymerases E arid and are part of a multiprotein DNA replication complex...a cyclin-dependent kinase inhibitor, p21WAF1 ability to inhibit DNA replication in response to DNA damage has been wall characterized. Interestingly

RAD51 and RTEL1 compensate telomere loss in the absence of telomerase

PubMed Central

Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon

2018-01-01

Abstract Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication. PMID:29346668

Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast.

PubMed

Contamine, V; Picard, M

2000-06-01

Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho(+) mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho(0) petites) and/or lead to truncated forms (rho(-)) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho(+) genome depends on a centromere-like structure dispensable for the maintenance of rho(-) mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.

Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch

PubMed Central

Hwang, Yung; Futran, Melinda; Hidalgo, Daniel; Pop, Ramona; Iyer, Divya Ramalingam; Scully, Ralph; Rhind, Nicholas; Socolovsky, Merav

2017-01-01

Cell cycle regulators are increasingly implicated in cell fate decisions, such as the acquisition or loss of pluripotency and self-renewal potential. The cell cycle mechanisms that regulate these cell fate decisions are largely unknown. We studied an S phase–dependent cell fate switch, in which murine early erythroid progenitors transition in vivo from a self-renewal state into a phase of active erythroid gene transcription and concurrent maturational cell divisions. We found that progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-renewal, a novel function for cyclin-dependent kinase inhibitors. The switch to differentiation entails rapid down-regulation of p57KIP2 with a consequent global increase in replication fork speed and an abruptly shorter S phase. Our work suggests that cell cycles with specialized global DNA replication dynamics are integral to the maintenance of specific cell states and to cell fate decisions. PMID:28560351

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress.

PubMed

Amaral, Nuno; Vendrell, Alexandre; Funaya, Charlotta; Idrissi, Fatima-Zahra; Maier, Michael; Kumar, Arun; Neurohr, Gabriel; Colomina, Neus; Torres-Rosell, Jordi; Geli, María-Isabel; Mendoza, Manuel

2016-05-01

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.

Fanconi anemia: causes and consequences of genetic instability.

PubMed

Kalb, R; Neveling, K; Nanda, I; Schindler, D; Hoehn, H

2006-01-01

Fanconi anemia (FA) is a rare recessive disease that reflects the cellular and phenotypic consequences of genetic instability: growth retardation, congenital malformations, bone marrow failure, high risk of neoplasia, and premature aging. At the cellular level, manifestations of genetic instability include chromosomal breakage, cell cycle disturbance, and increased somatic mutation rates. FA cells are exquisitely sensitive towards oxygen and alkylating drugs such as mitomycin C or diepoxybutane, pointing to a function of FA genes in the defense against reactive oxygen species and other DNA damaging agents. FA is caused by biallelic mutations in at least 12 different genes which appear to function in the maintenance of genomic stability. Eight of the FA proteins form a nuclear core complex with a catalytic function involving ubiquitination of the central FANCD2 protein. The posttranslational modification of FANCD2 promotes its accumulation in nuclear foci, together with known DNA maintenance proteins such as BRCA1, BRCA2, and the RAD51 recombinase. Biallelic mutations in BRCA2 cause a severe FA-like phenotype, as do biallelic mutations in FANCD2. In fact, only leaky or hypomorphic mutations in this central group of FA genes appear to be compatible with life birth and survival. The newly discovered FANCJ (= BRIP1) and FANCM (= Hef ) genes correspond to known DNA-maintenance genes (helicase resp. helicase-associated endonuclease for fork-structured DNA). These genes provide the most convincing evidence to date of a direct involvement of FA genes in DNA repair functions associated with the resolution of DNA crosslinks and stalled replication forks. Even though genetic instability caused by mutational inactivation of the FANC genes has detrimental effects for the majority of FA patients, around 20% of patients appear to benefit from genetic instability since genetic instability also increases the chance of somatic reversion of their constitutional mutations. Intragenic crossover, gene conversion, back mutation and compensating mutations in cis have all been observed in revertant, and, consequently, mosaic FA-patients, leading to improved bone marrow function. There probably is no other experiment of nature in our species in which causes and consequences of genetic instability, including the role of reactive oxygen species, can be better documented and explored than in FA.

Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration.

PubMed

Hafner, Lukas; Lezaja, Aleksandra; Zhang, Xu; Lemmens, Laure; Shyian, Maksym; Albert, Benjamin; Follonier, Cindy; Nunes, Jose Manuel; Lopes, Massimo; Shore, David; Mattarocci, Stefano

2018-04-24

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Cell culture-adaptive mutations of NS5A affect replication of hepatitis C virus differentially depending on the viral genotypes.

PubMed

Chung, Aeri; Jin, Bora; Han, Kwang-Hyub; Ahn, Sang Hoon; Kim, Seungtaek

2017-01-01

Most of HCV RNAs require cell culture-adaptive mutations for efficient replication in cell culture and a number of such mutations have been described including a well-known S2204I substitution mutation in NS5A protein. In contrast, the replication of genotype 2a JFH1 RNA in cell culture does not require any cell culture-adaptive mutation. Rather, the presence of S2204I mutation impaired the JFH1 RNA replication. In this study, we examined the effect of reversions and substitutions of NS5A cell culture-adaptive mutations on virus replication in different genotypic backgrounds after either placing genotype 1a NS5A in the genotype 2a JFH1 or vice versa. The results from this investigation suggest that the S2204I mutation affects HCV RNA replication differentially depending on the viral genotypes but that the effect was not simply explained by the genotypic background. Perhaps, the effect of the S2204I mutation on HCV replication reflects both intra- and intergenic interactions of NS5A protein. J. Med. Virol. 89:146-152, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Deficiency of the Arabidopsis Helicase RTEL1 Triggers a SOG1-Dependent Replication Checkpoint in Response to DNA Cross-Links

PubMed Central

Hu, Zhubing; Cools, Toon; Kalhorzadeh, Pooneh; Heyman, Jefri; De Veylder, Lieven

2015-01-01

To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint. PMID:25595823

Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism

PubMed Central

Özer, Özgün; Bhowmick, Rahul; Liu, Ying; Hickson, Ian D.

2018-01-01

Telomeres resemble common fragile sites (CFSs) in that they are difficult-to-replicate and exhibit fragility in mitosis in response to DNA replication stress. At CFSs, this fragility is associated with a delay in the completion of DNA replication until early mitosis, whereupon cells are proposed to switch to a RAD52-dependent form of break-induced replication. Here, we show that this mitotic DNA synthesis (MiDAS) is also a feature of human telomeres. Telomeric MiDAS is not restricted to those telomeres displaying overt fragility, and is a feature of a wide range of cell lines irrespective of whether their telomeres are maintained by telomerase or by the alternative lengthening of telomeres (ALT) mechanism. MiDAS at telomeres requires RAD52, and is mechanistically similar to CFS-associated MiDAS, with the notable exception that telomeric MiDAS does not require the MUS81-EME1 endonuclease. We propose a model whereby replication stress initiates a RAD52-dependent form of break-induced replication that bypasses a requirement for MUS81-EME1 to complete DNA synthesis in mitosis. PMID:29662610

Chromosome demise in the wake of ligase-deficient replication.

PubMed

Kouzminova, Elena A; Kuzminov, Andrei

2012-06-01

Bacterial DNA ligases, NAD⁺-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. © 2012 Blackwell Publishing Ltd.

Flipping the Switch from G1 to S Phase with E3 Ubiquitin Ligases

PubMed Central

Rizzardi, Lindsay F.

2012-01-01

The cell cycle ensures genome maintenance by coordinating the processes of DNA replication and chromosome segregation. Of particular importance is the irreversible transition from the G1 phase of the cell cycle to S phase. This transition marks the switch from preparing chromosomes for replication (“origin licensing”) to active DNA synthesis (“origin firing”). Ubiquitin-mediated proteolysis is essential for restricting DNA replication to only once per cell cycle and is the major mechanism regulating the G1 to S phase transition. Although some changes in protein levels are attributable to regulated mRNA abundance, protein degradation elicits very rapid changes in protein abundance and is critical for the sharp and irreversible transition from one cell cycle stage to the next. Not surprisingly, regulation of the G1-to-S phase transition is perturbed in most cancer cells, and deregulation of key molecular events in G1 and S phase drives not only cell proliferation but also genome instability. In this review we focus on the mechanisms by which E3 ubiquitin ligases control the irreversible transition from G1 to S phase in mammalian cells. PMID:23634252

Evolutionary dynamics of giant viruses and their virophages.

PubMed

Wodarz, Dominik

2013-07-01

Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested.

Evolutionary dynamics of giant viruses and their virophages

PubMed Central

Wodarz, Dominik

2013-01-01

Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested. PMID:23919155

Aneuploidy and asynchronous replication in non-alcholic fatty liver disease and cryptogenic cirrhosis.

PubMed

Laish, Ido; Mannasse-Green, Batya; Hadary, Ruth; Konikoff, Fred M; Amiel, Aliza; Kitay-Cohen, Yona

2016-11-15

Non-alcoholic fatty liver disease (NAFLD) and cryptogenic cirrhosis (CC), which is largely a late sequela of NAFLD, are considered pre-neoplastic conditions that might progress to hepatocellular carcinoma. Aneuploidy, telomere aggregates and synchronization of replication were evaluated as markers of genetic instability in these patients. Peripheral blood lymphocytes from 22 patients with NAFLD, 20 patients with CC and 20 age-matched healthy controls were analyzed. To determine random aneuploidy, we used the fluorescence in situ hybridization (FISH) with probes for chromosomes 9 and 18. The rate of aneuploidy was inferred from the fraction of cells revealing one, three or more hybridization signals per cell. Aggregate size was divided into three fusion groups of 2-5, 6-10 and 11-15 telomeres, relative to the size of a single telomere. The replication pattern was determined by FISH in two pairs of alleles, 15qter and 13qter. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. Significantly higher random aneuploidy rate was found in the CC patients than in the control group, and to a lesser degree in NAFLD patients. Telomere aggregates were insignificantly higher in both groups. Only patients with CC showed significantly higher rate of asynchronous replication with proportionately more cells with two single dots among the normal cells (p<0.001). These results likely reflect changes in gene replication and cell cycle progression in these conditions, possibly correlating with their malignant potential. Copyright © 2016 Elsevier B.V. All rights reserved.

Blocking Virus Replication during Acute Murine Cytomegalovirus Infection Paradoxically Prolongs Antigen Presentation and Increases the CD8+ T Cell Response by Preventing Type I IFN-Dependent Depletion of Dendritic Cells.

PubMed

Loo, Christopher P; Snyder, Christopher M; Hill, Ann B

2017-01-01

Increasing amounts of pathogen replication usually lead to a proportionate increase in size and effector differentiation of the CD8 + T cell response, which is attributed to increased Ag and inflammation. Using a murine CMV that is highly sensitive to the antiviral drug famciclovir to modulate virus replication, we found that increased virus replication drove increased effector CD8 + T cell differentiation, as expected. Paradoxically, however, increased virus replication dramatically decreased the size of the CD8 + T cell response to two immunodominant epitopes. The decreased response was due to type I IFN-dependent depletion of conventional dendritic cells and could be reproduced by specific depletion of dendritic cells from day 2 postinfection or by sterile induction of type I IFN. Increased virus replication and type I IFN specifically inhibited the response to two immunodominant epitopes that are known to be dependent on Ag cross-presented by DCs, but they did not inhibit the response to "inflationary" epitopes whose responses can be sustained by infected nonhematopoietic cells. Our results show that type I IFN can suppress CD8 + T cell responses to cross-presented Ag by depleting cross-presenting conventional dendritic cells. Copyright © 2016 by The American Association of Immunologists, Inc.

How can we model selectively neutral density dependence in evolutionary games.

PubMed

Argasinski, Krzysztof; Kozłowski, Jan

2008-03-01

The problem of density dependence appears in all approaches to the modelling of population dynamics. It is pertinent to classic models (i.e., Lotka-Volterra's), and also population genetics and game theoretical models related to the replicator dynamics. There is no density dependence in the classic formulation of replicator dynamics, which means that population size may grow to infinity. Therefore the question arises: How is unlimited population growth suppressed in frequency-dependent models? Two categories of solutions can be found in the literature. In the first, replicator dynamics is independent of background fitness. In the second type of solution, a multiplicative suppression coefficient is used, as in a logistic equation. Both approaches have disadvantages. The first one is incompatible with the methods of life history theory and basic probabilistic intuitions. The logistic type of suppression of per capita growth rate stops trajectories of selection when population size reaches the maximal value (carrying capacity); hence this method does not satisfy selective neutrality. To overcome these difficulties, we must explicitly consider turn-over of individuals dependent on mortality rate. This new approach leads to two interesting predictions. First, the equilibrium value of population size is lower than carrying capacity and depends on the mortality rate. Second, although the phase portrait of selection trajectories is the same as in density-independent replicator dynamics, pace of selection slows down when population size approaches equilibrium, and then remains constant and dependent on the rate of turn-over of individuals.

Modeling and Laboratory Investigations of Radiative Shocks

NASA Astrophysics Data System (ADS)

Grun, Jacob; Laming, J. Martin; Manka, Charles; Moore, Christopher; Jones, Ted; Tam, Daniel

2001-10-01

Supernova remnants are often inhomogeneous, with knots or clumps of material expanding in ambient plasma. This structure may be initiated by hydrodynamic instabilities occurring during the explosion, but it may plausibly be amplified by instabilities of the expanding shocks such as, for example, corrugation instabilities described by D’yakov in 1954, Vishniac in 1983, and observed in the laboratory by Grun et al. in 1991. Shock instability can occur when radiation lowers the effective adiabatic index of the gas. In view of the difficulty of modeling radiation in non-equilibrium plasmas, and the dependence of shock instabilities on such radiation, we are performing a laboratory experiment to study radiative shocks. The shocks are generated in a miniature, laser-driven shock tube. The gas density inside the tube at any instant in time is measured using time and space-resolved interferometry, and the emission spectrum of the gas is measured with time-resolved spectroscopy. We simulate the experiment with a 1D code that models time dependent post-shock ionization and non-equilibrium radiative cooling. S. P. D’yakov, Zhurnal Eksperimentalnoi Teoreticheskoi Fiziki 27, 288 (1954); see also section 90 in L.D. Landau and E.M. Lifshitz, Fluid Mechanics (Butterworth-Heinemann 1987); E.T. Vishniac, Astrophys. J. 236, 880 (1983); J. Grun, et al., Phys. Rev. Lett., 66, 2738 (1991)

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-Replication

DTIC Science & Technology

2005-04-01

with 50’C SCE (1 M sorbitol, 0.1 M Na Antcdc6p becomes undetectable within 30 mi after galac- citrate , and 10 mM EDTA). Lyticase was added to a final...and then placed in SCEM + lyticase [1 M sorbitol, decrease in colony-forming units after 3 h in galactose. In 0.1 M Na citrate , 10 mM EDTA, 5% j3...nocodazole, exposed to 20 /g/ml of the DNA damag- sponse triggered by rereplication. The nearly complete con- ing agent phleomycin, and examined by

Brefeldin A Inhibits Cell-Free, De Novo Synthesis of Poliovirus

PubMed Central

Cuconati, Andrea; Molla, Akhteruzzaman; Wimmer, Eckard

1998-01-01

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system. PMID:9658088

Fault stability under conditions of variable normal stress

USGS Publications Warehouse

Dieterich, J.H.; Linker, M.F.

1992-01-01

The stability of fault slip under conditions of varying normal stress is modelled as a spring and slider system with rate- and state-dependent friction. Coupling of normal stress to shear stress is achieved by inclining the spring at an angle, ??, to the sliding surface. Linear analysis yields two conditions for unstable slip. The first, of a type previously identified for constant normal stress systems, results in instability if stiffness is below a critical value. Critical stiffness depends on normal stress, constitutive parameters, characteristic sliding distance and the spring angle. Instability of the first type is possible only for velocity-weakening friction. The second condition yields instability if spring angle ?? <-cot-1??ss, where ??ss is steady-state sliding friction. The second condition can arise under conditions of velocity strengthening or weakening. Stability fields for finite perturbations are investigated by numerical simulation. -Authors

Developmental instability in Rhus copallinum L.: multiple stressors, years, and responses

USGS Publications Warehouse

Freeman, D. Carl; Brown, Michelle L.; Duda, Jeffrey J.; Graham, John H.; Emlen, John M.; Krzysik, Anthony J.; Balbach, Harold E.; Kovacic, Dave A.; Zak, John C.

2004-01-01

Developmental instability, as assessed by leaf fluctuating asymmetry and stem internode allometry, was examined at nine sites, representing three levels of disturbance, over multiple years. Site selection was based on land‐use disturbance classes related to training of mechanized infantry and other land management activities at Fort Benning, Georgia. Developmental instability varied among sites and years, and there was a strong site‐by‐year interaction for many traits. Indeed, depending on the year, the same site could be ranked as having the greatest and least amount of leaf fluctuating asymmetry. Burning a site the year prior to collecting the leaves profoundly influenced measures of leaf fluctuating asymmetry. In the absence of recent burning, leaf fluctuating asymmetry declined with increasing disturbance, but burning the year prior to collecting the leaves reversed this trend. Total plant cover, proportion of bare ground, and amount of plant litter influenced the amount of leaf asymmetry in a site‐dependent manner. Overall, burning influenced the levels of developmental instability more than either disturbance or microhabitat variables such as total plant cover, which should reflect competition in a plant’s immediate neighborhood.

Evaluation of Chromosomal Instability in Diabetic Rats Treated with Naringin

PubMed Central

A. Bakheet, Saleh; M. Attia, Sabry

2011-01-01

We used the bone marrow DNA strand breaks, micronucleus formations, spermatocyte chromosomal aberrations, and sperm characteristic assays to investigate the chromosomal instability in somatic and germinal cells of diabetic rats treated with multiple doses of naringin. The obtained results revealed that naringin was neither cytotoxic nor genotoxic for the rats at all tested doses. Moreover, naringin significantly reduced the diabetes-induced chromosomal instability in somatic and germinal cells in a dose-dependent manner. In addition, diabetes induced marked biochemical alterations characteristic of oxidative stress including enhanced lipid peroxidation, accumulation of oxidized glutathione, reduction in reduced glutathione, and accumulation of intracellular reactive oxygen species. Treatment with naringin ameliorated these biochemical markers dose-dependently. In conclusion, naringin confers an appealing protective effect against diabetes-induced chromosomal instability towards rat somatic and germinal cells which might be explained partially via diminishing the de novo free radical generation induced by hyperglycemia. Thus, naringin might be a good candidate to reduce genotoxic risk associated with hyperglycemia and may provide decreases in the development of secondary malignancy and abnormal reproductive outcomes risks, which seems especially important for diabetic patients. PMID:21941606

Sister acts: coordinating DNA replication and cohesion establishment

PubMed Central

Sherwood, Rebecca; Takahashi, Tatsuro S.; Jallepalli, Prasad V.

2010-01-01

The ring-shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesin's ability to generate cohesive linkages is restricted to S phase and depends on loading and establishment factors that are intimately connected to DNA replication. Here we review how cohesin is regulated by the replication machinery, as well as recent evidence that cohesin itself influences how chromosomes are replicated. PMID:21159813

BRCA1 is Required for Post-replication Repair After UV-induced DNA Damage

PubMed Central

Pathania, Shailja; Nguyen, Jenna; Hill, Sarah J.; Scully, Ralph; Feunteun, Jean; Livingston, David M.

2011-01-01

BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent, but nucleotide excision repair- independent manner. More specifically, at UV- stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and post- replicative repair. These BRCA1 functions differ from those required for DSBR. PMID:21963239

Instability analysis of expansion-free sphere in f(𝒢) gravity

NASA Astrophysics Data System (ADS)

Sharif, M.; Ikram, Ayesha

The aim of this paper is to study the dynamical instability of expansion-free spherically symmetric anisotropic fluid in the framework of f(𝒢) gravity. We apply perturbation scheme of the first-order to the metric functions as well as matter variables and construct modified field equations for both static and perturbed configurations using power-law f(𝒢) model. To discuss the instability dynamics, we use the contracted Bianchi identities to formulate the dynamical equations in both Newtonian and post-Newtonian regimes. It is found that the range of instability is independent of adiabatic index for expansion-free fluid but depends on anisotropic pressures, energy density and Gauss-Bonnet (GB) terms.

ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress

PubMed Central

Rodriguez, Jairo; Tsukiyama, Toshio

2013-01-01

Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks. PMID:23307868

Influence of nonlinear interactions on the development of instability in hydrodynamic wave systems

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

Romanova, N. N.; Chkhetiani, O. G., E-mail: ochkheti@mx.iki.rssi.ru, E-mail: ochkheti@gmail.ru; Yakushkin, I. G.

2016-05-15

The problem of the development of shear instability in a three-layer medium simulating the flow of a stratified incompressible fluid is considered. The hydrodynamic equations are solved by expanding the Hamiltonian in a small parameter. The equations for three interacting waves, one of which is unstable, have been derived and solved numerically. The three-wave interaction is shown to stabilize the instability. Various regimes of the system’s dynamics, including the stochastic ones dependent on one of the invariants in the problem, can arise in this case. It is pointed out that the instability development scenario considered differs from the previously consideredmore » scenario of a different type, where the three-wave interaction does not stabilize the instability. The interaction of wave packets is considered briefly.« less

Transient triggering of near and distant earthquakes

USGS Publications Warehouse

Gomberg, J.; Blanpied, M.L.; Beeler, N.M.

1997-01-01

We demonstrate qualitatively that frictional instability theory provides a context for understanding how earthquakes may be triggered by transient loads associated with seismic waves from near and distance earthquakes. We assume that earthquake triggering is a stick-slip process and test two hypotheses about the effect of transients on the timing of instabilities using a simple spring-slider model and a rate- and state-dependent friction constitutive law. A critical triggering threshold is implicit in such a model formulation. Our first hypothesis is that transient loads lead to clock advances; i.e., transients hasten the time of earthquakes that would have happened eventually due to constant background loading alone. Modeling results demonstrate that transient loads do lead to clock advances and that the triggered instabilities may occur after the transient has ceased (i.e., triggering may be delayed). These simple "clock-advance" models predict complex relationships between the triggering delay, the clock advance, and the transient characteristics. The triggering delay and the degree of clock advance both depend nonlinearly on when in the earthquake cycle the transient load is applied. This implies that the stress required to bring about failure does not depend linearly on loading time, even when the fault is loaded at a constant rate. The timing of instability also depends nonlinearly on the transient loading rate, faster rates more rapidly hastening instability. This implies that higher-frequency and/or longer-duration seismic waves should increase the amount of clock advance. These modeling results and simple calculations suggest that near (tens of kilometers) small/moderate earthquakes and remote (thousands of kilometers) earthquakes with magnitudes 2 to 3 units larger may be equally effective at triggering seismicity. Our second hypothesis is that some triggered seismicity represents earthquakes that would not have happened without the transient load (i.e., accumulated strain energy would have been relieved via other mechanisms). We test this using two "new-seismicity" models that (1) are inherently unstable but slide at steady-state conditions under the background load and (2) are conditionally stable such that instability occurs only for sufficiently large perturbations. For the new-seismicity models, very small-amplitude transients trigger instability relative to the clock-advance models. The unstable steady-state models predict that the triggering delay depends inversely and nonlinearly on the transient amplitude (as in the clock-advance models). We were unable to generate delayed triggering with conditionally stable models. For both new-seismicity models, the potential for triggering is independent of when the transient load is applied or, equivalently, of the prestress (unlike in the clock-advance models). In these models, a critical triggering threshold appears to be inversely proportional to frequency. Further advancement of our understanding will require more sophisticated, quantitative models and observations that distinguish between our qualitative, yet distinctly different, model predictions.

Hydrodynamic instabilities of flows involving melting in under-saturated porous media

NASA Astrophysics Data System (ADS)

Sajjadi, M.; Azaiez, J.

2016-03-01

The process of melting in partially saturated porous media is modeled for flow displacements prone to hydrodynamic instabilities due to adverse mobility ratios. The effects of the development of instabilities on the melting process are investigated through numerical simulations as well as analytical solution to unravel the physics of the flow. The effects of melting parameters, namely, the melting potential of the fluid, the rate of heat transfer to the frozen phase, and the saturation of the frozen material along with the parameters defining the viscous forces, i.e., the thermal and solutal log mobility ratios are examined. Results are presented for different scenarios and the enhancement or attenuation of instabilities are discussed based on the dominant physical mechanisms. Beside an extensive qualitative analysis, the performance of different displacement scenarios is compared with respect to the melt production and the extent of contribution of instability to the enhancement of melting. It is shown that the hydrodynamic instabilities tend in general to enhance melting but the rate of enhancement depends on the interplay between the instabilities and melting at the thermal front. A larger melting potential and a smaller saturation of the frozen material tend to increase the contribution of instability to melting.

Molecular analysis of the replication program in unicellular model organisms.

PubMed

Raghuraman, M K; Brewer, Bonita J

2010-01-01

Eukaryotes have long been reported to show temporal programs of replication, different portions of the genome being replicated at different times in S phase, with the added possibility of developmentally regulated changes in this pattern depending on species and cell type. Unicellular model organisms, primarily the budding yeast Saccharomyces cerevisiae, have been central to our current understanding of the mechanisms underlying the regulation of replication origins and the temporal program of replication in particular. But what exactly is a temporal program of replication, and how might it arise? In this article, we explore this question, drawing again on the wealth of experimental information in unicellular model organisms.

Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential

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

Dungey, Fiona A.; Loeser, Dana A.; Chalmers, Anthony J.

2008-11-15

Purpose: Current treatments for glioblastoma multiforme are inadequate and limited by the radiation sensitivity of normal brain. Because glioblastoma multiforme are rapidly proliferating tumors within nondividing normal tissue, the therapeutic ratio might be enhanced by combining radiotherapy with a replication-specific radiosensitizer. KU-0059436 (AZD2281) is a potent and nontoxic inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) undergoing a Phase II clinical trial as a single agent. Methods and Materials: Based on previous observations that the radiosensitizing effects of PARP inhibition are more pronounced in dividing cells, we investigated the mechanisms underlying radiosensitization of human glioma cells by KU-0059436, evaluating the replication dependence ofmore » this effect and its therapeutic potential. Results: KU-0059436 increased the radiosensitivity of four human glioma cell lines (T98G, U373-MG, UVW, and U87-MG). Radiosensitization was enhanced in populations synchronized in S phase and abrogated by concomitant exposure to aphidicolin. Sensitization was further enhanced when the inhibitor was combined with a fractionated radiation schedule. KU-0059436 delayed repair of radiation-induced DNA breaks and was associated with a replication-dependent increase in {gamma}H2AX and Rad51 foci. Conclusion: The results of our study have shown that KU-0059436 increases radiosensitivity in a replication-dependent manner that is enhanced by fractionation. A mechanism is proposed whereby PARP inhibition increases the incidence of collapsed replication forks after ionizing radiation, generating persistent DNA double-strand breaks. These observations indicate that KU-0059436 is likely to enhance the therapeutic ratio achieved by radiotherapy in the treatment of glioblastoma multiforme. A Phase I clinical trial is in development.« less

Particle Acceleration, Magnetic Field Generation, and Emission in Relativistic Pair Jets

NASA Technical Reports Server (NTRS)

Nishikawa, K.-I.; Ramirez-Ruiz, E.; Hardee, P.; Hededal, C.; Mizuno, Y.

2005-01-01

Shock acceleration is a ubiquitous phenomenon in astrophysical plasmas. Plasma waves and their associated instabilities (e.g., the Buneman instability, two-streaming instability, and the Weibel instability) created by relativistic pair jets are responsible for particle (electron, positron, and ion) acceleration. Using a 3-D relativistic electromagnetic particle (REMP) code, we have investigated particle acceleration associated with a relativistic jet propagating through an ambient plasma with and without initial magnetic fields. The growth rates of the Weibel instability depends on the distribution of pair jets. Simulations show that the Weibel instability created in the collisionless shock accelerates particles perpendicular and parallel to the jet propagation direction. The simulation results show that this instability is responsible for generating and amplifying highly nonuniform, small-scale magnetic fields, which contribute to the electron's transverse deflection behind the jet head. The "jitter" radiation from deflected electrons has different properties than synchrotron radiation which is calculated in a uniform magnetic field. This jitter radiation may be important to understanding the complex time evolution and/or spectral structure in gamma-ray bursts, relativistic jets, and supernova remnants.

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

PubMed Central

Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

2015-01-01

The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

PubMed Central

Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

2015-01-01

ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. PMID:26699641

Influence of heating rate on the condensational instability. [in outer layers of solar atmosphere

NASA Technical Reports Server (NTRS)

Dahlburg, R. B.; Mariska, J. T.

1988-01-01

Analysis and numerical simulation are used to determine the effect that various heating rates have on the linear and nonlinear evolution of a typical plasma within a solar magnetic flux tube subject to the condensational instability. It is found that linear stability depends strongly on the heating rate. The results of numerical simulations of the nonlinear evolution of the condensational instability in a solar magnetic flux tube are presented. Different heating rates lead to quite different nonlinear evolutions, as evidenced by the behavior of the global internal energy.

Dynamic stability of electrodynamic maglev systems

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

Cai, Y.; Chen, S.S.; Mulcahy, T.M.

1997-01-01

Because dynamic instabilities are not acceptable in any commercial maglev system, it is important to consider dynamic instability in the development of all maglev systems. This study considers the stability of maglev systems based on mathematical models and experimental data. Divergence and flutter are obtained for coupled vibration of a three-degree-of-freedom maglev vehicle on a guideway consisting of double L-shaped aluminum segments. The theory and analysis for motion-dependent magnetic-force-induced instability developed in this study provides basic stability characteristics and identifies future research needs for maglev systems.

Screech tones from free and ducted supersonic jets

NASA Technical Reports Server (NTRS)

Tam, C. K. W.; Ahuja, K. K.; Jones, R. R., III

1993-01-01

The dependence of the instability wave spectrum on azimuthal mode number, the jet to ambient gas temperature ratio, and the jet Mach number is studied. It is shown that the switch of the dominant screech mode (axisymmetric to helical/flapping) as Mach number increases is due to the switch in dominance of the corresponding mode of instability waves. Super-resonance can occur when the feedback loop is powered by the most amplified instability wave. It is suggested that the large amplitude pressure fluctuations and tone in the test cells are generated by super-resonance.

Universal instability of hairy black holes in Lovelock-Galileon theories in D dimensions

NASA Astrophysics Data System (ADS)

Takahashi, Kazufumi; Suyama, Teruaki; Kobayashi, Tsutomu

2016-03-01

We analyze spherically symmetric black hole solutions with time-dependent scalar hair in a class of Lovelock-Galileon theories, which are the scalar-tensor theories with second-order field equations in arbitrary dimensions. We first show that known black hole solutions in five dimensions are always plagued by the ghost/gradient instability in the vicinity of the horizon. We then generalize such black hole solutions to higher dimensions and show that the same instability found in five dimensions appears universally in any number of dimensions.

Noise-induced bistability in the quasi-neutral coexistence of viral RNAs under different replication modes.

PubMed

Sardanyés, Josep; Arderiu, Andreu; Elena, Santiago F; Alarcón, Tomás

2018-05-01

Evolutionary and dynamical investigations into real viral populations indicate that RNA replication can range between the two extremes represented by so-called 'stamping machine replication' (SMR) and 'geometric replication' (GR). The impact of asymmetries in replication for single-stranded (+) sense RNA viruses has been mainly studied with deterministic models. However, viral replication should be better described by including stochasticity, as the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasi-neutral coexistence scenario, with a line of fixed points involving different strands' equilibrium ratios depending on the initial conditions. Recent research into the quasi-neutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alter the mean-field scenario, and one of the two species outcompetes the other. In this article, we study this phenomenon for viral RNA replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNAs, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication. © 2018 The Author(s).

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

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

Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatiblemore » with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.« less

Interpreting the Dependence of Mutation Rates on Age and Time

PubMed Central

Gao, Ziyue; Wyman, Minyoung J.; Sella, Guy; Przeworski, Molly

2016-01-01

Mutations can originate from the chance misincorporation of nucleotides during DNA replication or from DNA lesions that arise between replication cycles and are not repaired correctly. We introduce a model that relates the source of mutations to their accumulation with cell divisions, providing a framework for understanding how mutation rates depend on sex, age, and cell division rate. We show that the accrual of mutations should track cell divisions not only when mutations are replicative in origin but also when they are non-replicative and repaired efficiently. One implication is that observations from diverse fields that to date have been interpreted as pointing to a replicative origin of most mutations could instead reflect the accumulation of mutations arising from endogenous reactions or exogenous mutagens. We further find that only mutations that arise from inefficiently repaired lesions will accrue according to absolute time; thus, unless life history traits co-vary, the phylogenetic “molecular clock” should not be expected to run steadily across species. PMID:26761240

IFN-Dependent and -Independent Reduction in West Nile Virus Infectivity in Human Dermal Fibroblasts

PubMed Central

Hoover, Lisa I.; Fredericksen, Brenda L.

2014-01-01

Although dermal fibroblasts are one of the first cell types exposed to West Nile virus (WNV) during a blood meal by an infected mosquito, little is known about WNV replication within this cell type. Here, we demonstrate that neuroinvasive, WNV-New York (WNV-NY), and nonneuroinvasive, WNV-Australia (WNV-AUS60) strains are able to infect and replicate in primary human dermal fibroblasts (HDFs). However, WNV-AUS60 replication and spread within HDFs was reduced compared to that of WNV-NY due to an interferon (IFN)-independent reduction in viral infectivity early in infection. Additionally, replication of both strains was constrained late in infection by an IFN-β-dependent reduction in particle infectivity. Overall, our data indicates that human dermal fibroblasts are capable of supporting WNV replication; however, the low infectivity of particles produced from HDFs late in infection suggests that this cell type likely plays a limited role as a viral reservoir in vivo. PMID:24662674

N-body simulations of viscous instability of planetary rings

NASA Astrophysics Data System (ADS)

Salo, Heikki; Schmidt, Jürgen

2010-04-01

We study viscous instability of planetary rings in terms of N-body simulations. We show that for rings composed of fairly elastic particles (e.g. as in Hatzes et al. [Hatzes, A., Bridges, F.G., Lin, D.N.C., 1988. Collisional properties of ice spheres at low impact velocities. Mon. Not. R. Astron. Soc. 231, 1091-1115]) the instability may lead to the spontaneous formation of dense ringlets in a background of lower density. In most parts of Saturn's rings the particle collisions are probably much more dissipative, as suggested by the presence of self-gravity wakes, and classic viscous instability should be suppressed. However, our results demonstrate that the mechanism of viscous instability itself is valid. The dynamical effects of size-dependent elasticity in a system with a size distribution have never been studied before. We show that this may in principle lead to a size-selective viscous instability, small particles concentrating on ringlets against the more uniform background of large particles.

On the marginal instability threshold condition of the aperiodic ordinary mode

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

Schlickeiser, R.; Yoon, P. H.; School of Space Research, Kyung Hee University, Yongin

2014-07-15

The purely growing ordinary (O) mode instability has recently received renewed attention owing to its potential applicability to the solar wind plasma. Here, an analytical marginal instability condition is derived for counter-streaming bi-Maxwellian plasma particle distribution functions. The derived marginal instability condition as a function of the temperature anisotropy and plasma beta agrees remarkably well with the numerically determined instability condition. The existence of a new instability domain of the O-mode at small plasma beta values is confirmed with the leading A∝β{sub ∥}{sup −1}-dependence, if the counter-stream parameter P{sub e} exceeds a critical value. At small plasma beta values atmore » large enough counter-stream parameter, the O-mode also operates for temperature anisotropies A = T{sub ⊥}/T{sub ∥} > 1 even larger than unity, as the parallel counter-stream free energy exceeds the perpendicular bi-Maxwellian free energy.« less

Elliptical Instability of Rotating Von Karman Street

NASA Astrophysics Data System (ADS)

Stegner, A.; Pichon, T.; Beunier, M.

Clouds often reveal a meso-scale vortex shedding in the wake of mountainous islands. Unlike the classical bi-dimensional Von-Karman street, these observed vortex street are affected by the earth rot ation and vertical stratification. Theses effects could induce a selective destabilization of anticyclonic vortices. It is well known that inertial instability (also called centrifugal instability) induce a three- dimensional destabilization of anticyclonic structures when the absolute vorticity is larger than the local Coriolis parameter. However, we have shown, by the mean of laboratory experiments, that it is a different type of instability which is mainly responsible for asymmetric rotating Von-Karman street. A serie of experiments were performed to study the wake of a cylinder in a rotating fluid, at medium Reynolds number and order one Rossby number. We have shown that the vertical structure of unstable anticyclonic vortices is characteristic of an elliptical instability. Besides, unlike the inertial instability, the vertical unstable wavelength depends on the Rossby number.

Interfacial fluid instabilities and Kapitsa pendula.

PubMed

Krieger, Madison S

2017-07-01

The onset and development of instabilities is one of the central problems in fluid mechanics. Here we develop a connection between instabilities of free fluid interfaces and inverted pendula. When acted upon solely by the gravitational force, the inverted pendulum is unstable. This position can be stabilized by the Kapitsa phenomenon, in which high-frequency low-amplitude vertical vibrations of the base creates a fictitious force which opposes the gravitational force. By transforming the dynamical equations governing a fluid interface into an appropriate pendulum-type equation, we demonstrate how stability can be induced in fluid systems by properly tuned vibrations. We construct a "dictionary"-type relationship between various pendula and the classical Rayleigh-Taylor, Kelvin-Helmholtz, Rayleigh-Plateau and the self-gravitational instabilities. This makes several results in control theory and dynamical systems directly applicable to the study of tunable fluid instabilities, where the critical wavelength depends on the external forces or the instability is suppressed entirely. We suggest some applications and instances of the effect ranging in scale from microns to the radius of a galaxy.

Secondary Instability of Stationary Crossflow Vortices in Mach 6 Boundary Layer Over a Circular Cone

NASA Technical Reports Server (NTRS)

Li, Fei; Choudhari, Meelan M.; Paredes-Gonzalez, Pedro; Duan, Lian

2015-01-01

Hypersonic boundary layer flows over a circular cone at moderate incidence can support strong crossflow instability. Due to more efficient excitation of stationary crossflow vortices by surface roughness, such boundary layer flows may transition to turbulence via rapid amplification of the high-frequency secondary instabilities of finite amplitude stationary crossflow vortices. The amplification characteristics of these secondary instabilities are investigated for crossflow vortices generated by an azimuthally periodic array of roughness elements over a 7-degree half-angle circular cone in a Mach 6 free stream. Depending on the local amplitude of the stationary crossflow mode, the most unstable secondary disturbances either originate from the second (i.e., Mack) mode instabilities of the unperturbed boundary layer or correspond to genuine secondary instabilities that reduce to stable disturbances at sufficiently small amplitudes of the stationary crossflow vortex. The predicted frequencies of dominant secondary disturbances are similar to those measured during wind tunnel experiments at Purdue University and the Technical University of Braunschweig, Germany.

The Multivariate Roles of Family Instability and Interparental Conflict in Predicting Children's Representations of Insecurity in the Family System and Early School Adjustment Problems.

PubMed

Coe, Jesse L; Davies, Patrick T; Sturge-Apple, Melissa L

2017-02-01

This study examined the moderating role of family instability in relations involving destructive interparental conflict, children's internal representations of insecurity in the family system, and their early school maladjustment. Two hundred forty-three preschool children (M age = 4.60 years; 56 % girls) and their families participated in this multi-method (i.e., observations, structured interview, surveys) multi-informant (i.e., observer, parent, teacher), longitudinal study. Findings indicated that the mediational role of children's insecure family representations in the pathway between destructive interparental conflict and children's adjustment problems varied significantly depending on the level of family instability. Interparental conflict was specifically associated with insecure family representations only under conditions of low family instability. In supporting the role of family instability as a vulnerable-stable risk factor, follow up analyses revealed that children's concerns about security in the family were uniformly high under conditions of heightened instability regardless of their level of exposure to interparental conflict.

Kinetic instability of electrostatic ion cyclotron waves in inter-penetrating plasmas

NASA Astrophysics Data System (ADS)

Bashir, M. F.; Ilie, R.; Murtaza, G.

2018-05-01

The Electrostatic Ion Cyclotron (EIC) instability that includes the effect of wave-particle interaction is studied owing to the free energy source through the flowing velocity of the inter-penetrating plasmas. It is shown that the origin of this current-less instability is different from the classical current driven EIC instability. The threshold conditions applicable to a wide range of plasma parameters and the estimate of the growth rate are determined as a function of the normalized flowing velocity ( u0/vt f e ), the temperature ( Tf/Ts ) and the density ratios ( nf 0/ns 0 ) of flowing component to static one. The EIC instability is driven by either flowing electrons or flowing ions, depending upon the different Doppler shifted frequency domains. It is found that the growth rate for electron-driven instability is higher than the ion-driven one. However, in both cases, the denser (hotter) is the flowing plasma, the lesser (greater) is the growth rate. The possible applications related to the terrestrial solar plasma environment are also discussed.

Animal models for posttraumatic stress disorder: An overview of what is used in research

PubMed Central

Borghans, Bart; Homberg, Judith R

2015-01-01

Posttraumatic stress disorder (PTSD) is a common anxiety disorder characterised by its persistence of symptoms after a traumatic experience. Although some patients can be cured, many do not benefit enough from the psychological therapies or medication strategies used. Many researchers use animal models to learn more about the disorder and several models are available. The most-used physical stressor models are single-prolonged stress, restraint stress, foot shock, stress-enhanced fear learning, and underwater trauma. Common social stressors are housing instability, social instability, early-life stress, and social defeat. Psychological models are not as diverse and rely on controlled exposure to the test animal’s natural predator. While validation of these models has been resolved with replicated symptoms using analogous stressors, translating new findings to human patients remains essential for their impact on the field. Choosing a model to experiment with can be challenging; this overview of what is possible with individual models may aid in making a decision. PMID:26740930

An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

PubMed

Liu, Pengfei; Yuan, Bo; Carvalho, Claudia M B; Wuster, Arthur; Walter, Klaudia; Zhang, Ling; Gambin, Tomasz; Chong, Zechen; Campbell, Ian M; Coban Akdemir, Zeynep; Gelowani, Violet; Writzl, Karin; Bacino, Carlos A; Lindsay, Sarah J; Withers, Marjorie; Gonzaga-Jauregui, Claudia; Wiszniewska, Joanna; Scull, Jennifer; Stankiewicz, Paweł; Jhangiani, Shalini N; Muzny, Donna M; Zhang, Feng; Chen, Ken; Gibbs, Richard A; Rautenstrauss, Bernd; Cheung, Sau Wai; Smith, Janice; Breman, Amy; Shaw, Chad A; Patel, Ankita; Hurles, Matthew E; Lupski, James R

2017-02-23

De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Single-stranded DNA binding protein Gp5 of Bacillus subtilis phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion.

PubMed

Tone, Takahiro; Takeuchi, Ari; Makino, Osamu

2012-01-01

In the absence of viral single-stranded DNA binding protein gp5, Bacillus subtilis phage φ29 failed to grow and to replicate its genome at 45 °C, while it grew and replicated normally at 30 °C and 42 °C. This indicates that gp5 is dispensable for φ29 DNA replication at 42 °C and lower temperatures.

Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

PubMed

Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

2014-01-01

DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. © 2013 John Wiley & Sons Ltd.

Self-replication: Nanostructure evolution

NASA Astrophysics Data System (ADS)

Simmel, Friedrich C.

2017-10-01

DNA origami nanostructures were utilized to replicate a seed pattern that resulted in the growth of populations of nanostructures. Exponential growth could be controlled by environmental conditions depending on the preferential requirements of each population.

Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

PubMed Central

Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

2015-01-01

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID. PMID:26263206

Tigers on trails: occupancy modeling for cluster sampling.

PubMed

Hines, J E; Nichols, J D; Royle, J A; MacKenzie, D I; Gopalaswamy, A M; Kumar, N Samba; Karanth, K U

2010-07-01

Occupancy modeling focuses on inference about the distribution of organisms over space, using temporal or spatial replication to allow inference about the detection process. Inference based on spatial replication strictly requires that replicates be selected randomly and with replacement, but the importance of these design requirements is not well understood. This paper focuses on an increasingly popular sampling design based on spatial replicates that are not selected randomly and that are expected to exhibit Markovian dependence. We develop two new occupancy models for data collected under this sort of design, one based on an underlying Markov model for spatial dependence and the other based on a trap response model with Markovian detections. We then simulated data under the model for Markovian spatial dependence and fit the data to standard occupancy models and to the two new models. Bias of occupancy estimates was substantial for the standard models, smaller for the new trap response model, and negligible for the new spatial process model. We also fit these models to data from a large-scale tiger occupancy survey recently conducted in Karnataka State, southwestern India. In addition to providing evidence of a positive relationship between tiger occupancy and habitat, model selection statistics and estimates strongly supported the use of the model with Markovian spatial dependence. This new model provides another tool for the decomposition of the detection process, which is sometimes needed for proper estimation and which may also permit interesting biological inferences. In addition to designs employing spatial replication, we note the likely existence of temporal Markovian dependence in many designs using temporal replication. The models developed here will be useful either directly, or with minor extensions, for these designs as well. We believe that these new models represent important additions to the suite of modeling tools now available for occupancy estimation in conservation monitoring. More generally, this work represents a contribution to the topic of cluster sampling for situations in which there is a need for specific modeling (e.g., reflecting dependence) for the distribution of the variable(s) of interest among subunits.

Instabilities of thin layers of conducting fluids produced by time dependent magnetic fields

NASA Astrophysics Data System (ADS)

Burguete, Javier

2011-11-01

We present the recent results of an experiment where thin layers of conducting fluids are forced by time-dependent magnetic fields perpendicular to their surface. We use as conducting fluid an In-Ga-Sn alloy, immersed in a 5% hydrocloric acid solution to prevent oxidation. The conducting layers have a circular shape, and are placed inside a set-up that produces the vertical magnetic field. Due to MHD effects, the competition between the Lorentz force and gravity triggers an instability of the free surface. The shape of this surface can adopt many different configurations, with a very rich dynamics, presenting azimuthal wave numbers between 3 and 8 for the explored parameters. The magnetic field evolves harmonically with a frequency up to 10Hz, small enough to not to observe skin depth effects and with a magnitude up to 0.1 T. Different resonant regions have been observed, for narrow windows of the forcing frequency. We have analysed the existence of thresholds for these instabilities, depending on the wave number and experimental parameters. These results are compared with others present in the literature.

Peeling-angle dependence of the stick-slip instability during adhesive tape peeling.

PubMed

Dalbe, Marie-Julie; Santucci, Stéphane; Vanel, Loïc; Cortet, Pierre-Philippe

2014-12-28

The influence of peeling angle on the dynamics observed during the stick-slip peeling of an adhesive tape has been investigated. This study relies on a new experimental setup for peeling at a constant driving velocity while keeping constant the peeling angle and peeled tape length. The thresholds of the instability are shown to be associated with a subcritical bifurcation and bistability of the system. The velocity onset of the instability is moreover revealed to strongly depend on the peeling angle. This could be the consequence of peeling angle dependance of either the fracture energy of the adhesive-substrate joint or the effective stiffness at play between the peeling front and the point at which the peeling is enforced. The shape of the peeling front velocity fluctuations is finally shown to progressively change from typical stick-slip relaxation oscillations to nearly sinusoidal oscillations as the peeling angle is increased. We suggest that this transition might be controlled by inertial effects possibly associated with the propagation of the peeling force fluctuations through elongation waves in the peeled tape.

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

Akhrymuk, Ivan; Frolov, Ilya; Frolova, Elena I., E-mail: evfrolova@UAB.edu

Alphaviruses are a family of positive-strand RNA viruses that circulate on all continents between mosquito vectors and vertebrate hosts. Despite a significant public health threat, their biology is not sufficiently investigated, and the mechanisms of alphavirus replication and virus–host interaction are insufficiently understood. In this study, we have applied a variety of experimental systems to further understand the mechanism by which infected cells detect replicating alphaviruses. Our new data strongly suggest that activation of the antiviral response by alphavirus-infected cells is determined by the integrity of viral genes encoding proteins with nuclear functions, and by the presence of two cellularmore » pattern recognition receptors (PRRs), RIG-I and MDA5. No type I IFN response is induced in their absence. The presence of either of these PRRs is sufficient for detecting virus replication. However, type I IFN activation in response to pathogenic alphaviruses depends on the basal levels of RIG-I or MDA5. - Highlights: • Both RIG-I and MDA5 detect alphavirus replication. • Alphavirus-induced transcriptional shutoff affects type I IFN induction. • Sensing of alphavirus replication by RIG-I and MDA5 depends on their concentrations. • High basal level of RIG-I and MDA5 allows IFN induction by pathogenic alphaviruses. • This dependence determines the discrepancy between the in vivo and in vitro data.« less

IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system.

PubMed

Bardill, J Patrick; Miller, Jennifer L; Vogel, Joseph P

2005-04-01

Legionella pneumophila replicates inside alveolar macrophages and causes an acute, potentially fatal pneumonia called Legionnaires' disease. The ability of this bacterium to grow inside of macrophages is dependent on the presence of a functional dot/icm type IV secretion system (T4SS). Proteins secreted by the Dot/Icm T4SS are presumed to alter the host endocytic pathway, allowing L. pneumophila to establish a replicative niche within the host cell. Here we show that a member of the SidE family of proteins interacts with IcmS and is required for full virulence in the protozoan host Acanthamoeba castellanii. Using immunofluorescence microscopy and adenylate cyclase fusions, we show that SdeA is secreted into host cells by L. pneumophila in an IcmS-dependent manner. The SidE-like proteins are secreted very early during macrophage infection, suggesting that they are important in the initial formation of the replicative phagosome. Secreted SidE family members show a similar localization to other Dot/Icm substrates, specifically, to the poles of the replicative phagosome. This common localization of secreted substrates of the Dot/Icm system may indicate the formation of a multiprotein complex on the cytoplasmic face of the replicative phagosome.

Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

PubMed Central

Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

2014-01-01

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

Roles of SLX1–SLX4, MUS81–EME1, and GEN1 in avoiding genome instability and mitotic catastrophe

PubMed Central

Sarbajna, Shriparna; Davies, Derek; West, Stephen C.

2014-01-01

The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM–TopoIIIα–RMI1–RMI2 (BTR complex), SLX1–SLX4–MUS81–EME1 (SLX–MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX–MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX–MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1–SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion. These observations demonstrate that the SLX1–SLX4 nuclease and the SLX4 scaffold play divergent roles in the maintenance of genome integrity in human cells. PMID:24831703

Deficiency of the Arabidopsis helicase RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA cross-links.

PubMed

Hu, Zhubing; Cools, Toon; Kalhorzadeh, Pooneh; Heyman, Jefri; De Veylder, Lieven

2015-01-01

To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint. © 2015 American Society of Plant Biologists. All rights reserved.

Some effects of horizontal discretization on linear baroclinic and symmetric instabilities

NASA Astrophysics Data System (ADS)

Barham, William; Bachman, Scott; Grooms, Ian

2018-05-01

The effects of horizontal discretization on linear baroclinic and symmetric instabilities are investigated by analyzing the behavior of the hydrostatic Eady problem in ocean models on the B and C grids. On the C grid a spurious baroclinic instability appears at small wavelengths. This instability does not disappear as the grid scale decreases; instead, it simply moves to smaller horizontal scales. The peak growth rate of the spurious instability is independent of the grid scale as the latter decreases. It is equal to cf /√{Ri} where Ri is the balanced Richardson number, f is the Coriolis parameter, and c is a nondimensional constant that depends on the Richardson number. As the Richardson number increases c increases towards an upper bound of approximately 1/2; for large Richardson numbers the spurious instability is faster than the Eady instability. To suppress the spurious instability it is recommended to use fourth-order centered tracer advection along with biharmonic viscosity and diffusion with coefficients (Δx) 4 f /(32√{Ri}) or larger where Δx is the grid scale. On the B grid, the growth rates of baroclinic and symmetric instabilities are too small, and converge upwards towards the correct values as the grid scale decreases; no spurious instabilities are observed. In B grid models at eddy-permitting resolution, the reduced growth rate of baroclinic instability may contribute to partially-resolved eddies being too weak. On the C grid the growth rate of symmetric instability is better (larger) than on the B grid, and converges upwards towards the correct value as the grid scale decreases.

Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

PubMed Central

Verma, Subhash C.; Lu, Jie; Cai, Qiliang; Kosiyatrakul, Settapong; McDowell, Maria E.; Schildkraut, Carl L.; Robertson, Erle S.

2011-01-01

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences. PMID:22072974

Outcomes of a rapid refeeding protocol in Adolescent Anorexia Nervosa.

PubMed

Madden, Sloane; Miskovic-Wheatley, Jane; Clarke, Simon; Touyz, Stephen; Hay, Phillipa; Kohn, Michael R

2015-01-01

The impact of severe malnutrition and medical instability in adolescent Anorexia Nervosa (AN) on immediate health and long-term development underscores the need for safe and efficient methods of refeeding. Current refeeding guidelines in AN advocate low initial caloric intake with slow increases in energy intake to avoid refeeding syndrome. This study demonstrates the potential for more rapid refeeding to promote initial weight recovery and correct medical instability in adolescent AN. Seventy-eight adolescents with AN (12-18 years), hospitalised in two specialist paediatric eating disorder units, for medical instability (bradycardia, hypotension, hypothermia, orthostatic instability and/or cardiac arrhythmia) were followed during a 2.5 week admission. Patients were refed using a standardised protocol commencing with 24-72 hours of continuous nasogastric feeds (ceased with daytime medical stability) and routine oral phosphate supplementation, followed by nocturnal feeds and a meal plan of 1200-2400 kcal/day aiming for a total caloric intake of 2400-3000 kcal/day. Along with indicators of medical stability, weight, phosphate and glucose levels were recorded. All patients gained weight in week one (M = 2.79 kg, SD = 1.27 kg) and at subsequent measurement points with an average gain of 5.12 kg (SD = 2.96) at 2.5 weeks. No patient developed hypophosphatemia, hypoglycaemia, or stigmata of the refeeding syndrome. The refeeding protocol resulted in immediate weight gain and was well tolerated with no indicators of refeeding syndrome. There were no significant differences in outcomes between the treatment sites, suggesting the protocol is replicable. Australian Clinical Trials Register number: ACTRN012607000009415.

Navigating yeast genome maintenance with functional genomics.

PubMed

Measday, Vivien; Stirling, Peter C

2016-03-01

Maintenance of genome integrity is a fundamental requirement of all organisms. To address this, organisms have evolved extremely faithful modes of replication, DNA repair and chromosome segregation to combat the deleterious effects of an unstable genome. Nonetheless, a small amount of genome instability is the driver of evolutionary change and adaptation, and thus a low level of instability is permitted in populations. While defects in genome maintenance almost invariably reduce fitness in the short term, they can create an environment where beneficial mutations are more likely to occur. The importance of this fact is clearest in the development of human cancer, where genome instability is a well-established enabling characteristic of carcinogenesis. This raises the crucial question: what are the cellular pathways that promote genome maintenance and what are their mechanisms? Work in model organisms, in particular the yeast Saccharomyces cerevisiae, has provided the global foundations of genome maintenance mechanisms in eukaryotes. The development of pioneering genomic tools inS. cerevisiae, such as the systematic creation of mutants in all nonessential and essential genes, has enabled whole-genome approaches to identifying genes with roles in genome maintenance. Here, we review the extensive whole-genome approaches taken in yeast, with an emphasis on functional genomic screens, to understand the genetic basis of genome instability, highlighting a range of genetic and cytological screening modalities. By revealing the biological pathways and processes regulating genome integrity, these analyses contribute to the systems-level map of the yeast cell and inform studies of human disease, especially cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Replication licensing and the DNA damage checkpoint

PubMed Central

Cook, Jeanette Gowen

2011-01-01

Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings suggest that checkpoints and replication are more intimately associated than previously appreciated, even in the absence of exogenous DNA damage. This review summarizes some of these developments. PMID:19482602

The control of paramyxovirus genome hexamer length and mRNA editing.

PubMed

Matsumoto, Yusuke; Ohta, Keisuke; Kolakofsky, Daniel; Nishio, Machiko

2018-04-01

The unusual ability of a human parainfluenza virus type 2 (hPIV2) nucleoprotein point mutation (NP Q202A ) to strongly enhance minigenome replication was found to depend on the absence of a functional, internal element of the bipartite replication promoter (CRII). This point mutation allows relatively robust CRII-minus minigenome replication in a CRII-independent manner, under conditions in which NP wt is essentially inactive. The nature of the amino acid at position 202 apparently controls whether viral RNA-dependent RNA polymerase (vRdRp) can, or cannot, initiate RNA synthesis in a CRII-independent manner. By repressing genome synthesis when vRdRp cannot correctly interact with CRII, gln 202 of N, the only residue of the RNA-binding groove that contacts a nucleotide base in the N-RNA, acts as a gatekeeper for wild-type (CRII-dependent) RNA synthesis. This ensures that only hexamer-length genomes are replicated, and that the critical hexamer phase of the cis -acting mRNA editing sequence is maintained. © 2018 Matsumoto et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Chromosomal instability drives metastasis through a cytosolic DNA response.

PubMed

Bakhoum, Samuel F; Ngo, Bryan; Laughney, Ashley M; Cavallo, Julie-Ann; Murphy, Charles J; Ly, Peter; Shah, Pragya; Sriram, Roshan K; Watkins, Thomas B K; Taunk, Neil K; Duran, Mercedes; Pauli, Chantal; Shaw, Christine; Chadalavada, Kalyani; Rajasekhar, Vinagolu K; Genovese, Giulio; Venkatesan, Subramanian; Birkbak, Nicolai J; McGranahan, Nicholas; Lundquist, Mark; LaPlant, Quincey; Healey, John H; Elemento, Olivier; Chung, Christine H; Lee, Nancy Y; Imielenski, Marcin; Nanjangud, Gouri; Pe'er, Dana; Cleveland, Don W; Powell, Simon N; Lammerding, Jan; Swanton, Charles; Cantley, Lewis C

2018-01-25

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Enhancement of particle-induced viscous fingering in bidisperse suspensions

NASA Astrophysics Data System (ADS)

Xu, Feng; Lee, Sungyon

2017-11-01

The novel particle-induced fingering instability is observed when bidisperse particle suspensions displace air in a Hele-Shaw cell. Leading to the instability, we observe that larger particles consistently enrich the fluid-fluid interface at a faster rate than the small particles. This size-dependent enrichment of the interface leads to an earlier onset of the fingering instability for bidisperse suspensions, compared to their monodisperse counterpart. Careful experiments are carried out by either systematically varying the ratio of large to small particles at fixed total concentrations, or by changing the total concentrations while the large particle concentrations are held constant. Experimental results show that the presence of large particle causes the instability to occur at concentrations as much as 5% lower than the pure small particle case. We also discuss the physical mechanism that drives the enrichment and the subsequent instability based on the modified suspension balance model.

The nature of interfacial instabilities in liquid metal batteries in a vertical magnetic field

NASA Astrophysics Data System (ADS)

Molokov, S.

2018-02-01

The nature of instabilities in liquid metal batteries in the presence of a vertical magnetic field has been studied. The battery consists of two liquid metal layers, connected to the collectors, and a layer with an electrolyte inbetween. The closed geometry in the horizontal plane has been replaced by a half-plane to get a better insight into the problem. As in aluminium reduction cells the instability is generated at the electrically insulating sidewall of the battery. A travelling-wave solution has been obtained, which shows that there are two modes of the instability, fast and slow. Either of these modes may be most unstable depending on the values of various parameters, the most important of which are the well-known parameter of the instability, β, and the density of the electrolyte. For the intermediate range of the electrolyte density the medium-size batteries may be expected to be stable.

Molecular Mechanisms of Radiation-Induced Genomic Instability in Human Cells

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

Howard L. Liber; Jeffrey L. Schwartz

2005-10-31

There are many different model systems that have been used to study chromosome instability. What is clear from all these studies is that conclusions concerning chromosome instability depend greatly on the model system and instability endpoint that is studied. The model system for our studies was the human B-lymphoblastoid cell line TK6. TK6 was isolated from a spontaneously immortalized lymphoblast culture. Thus there was no outside genetic manipulation used to immortalize them. TK6 is a relatively stable p53-normal immortal cell line (37). It shows low gene and chromosome mutation frequencies (19;28;31). Our general approach to studying instability in TK6 cellsmore » has been to isolate individual clones and analyze gene and chromosome mutation frequencies in each. This approach maximizes the possibility of detecting low frequency events that might be selected against in mass cultures.« less

Opposite Roles for p38MAPK-Driven Responses and Reactive Oxygen Species in the Persistence and Resolution of Radiation-Induced Genomic Instability

PubMed Central

Werner, Erica; Wang, Huichen; Doetsch, Paul W.

2014-01-01

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as 56Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a threshold-like response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization. PMID:25271419

Solar wind interaction with dusty plasmas produces instabilities and solitary structures

NASA Astrophysics Data System (ADS)

Saleem, H.; Ali, S.

2017-12-01

It is pointed out that the solar wind interaction with dusty magnetospheres of the planets can give rise to purely growing instabilities as well as nonlinear electric field structures. Linear dispersion relation of the low frequency electrostatic ion-acoustic wave (IAW) is modified in the presence of stationary dust and its frequency becomes larger than its frequency in usual electron ion plasma even if ion temperature is equal to the electron temperature. This dust-ion-acoustic wave (DIAW) either becomes a purely growing electrostatic instability or turns out to be the modified dust-ion-acoustic wave (mDIAW) depending upon the magnitude of shear flow scale length and its direction. Growth rate of shear flow-driven electrostatic instability in a plasma having negatively charged stationary dust is larger than the usual D'Angelo instability of electron-ion plasma. It is shown that shear modified dust ion acoustic wave (mDIAW) produces electrostatic solitons in the nonlinear regime. The fluid theory predicts the existence of electrostatic solitons in the dusty plasmas in those regions where the inhomogeneous solar wind flow is parallel to the planetary or cometary magnetic field lines. The amplitude and width of the solitary structure depends upon dust density and magnitude of shear in the flow. This is a general theoretical model which is applied to dusty plasma of Saturn's F-ring for illustration.

Statistical simulation of the magnetorotational dynamo.

PubMed

Squire, J; Bhattacharjee, A

2015-02-27

Turbulence and dynamo induced by the magnetorotational instability (MRI) are analyzed using quasilinear statistical simulation methods. It is found that homogenous turbulence is unstable to a large-scale dynamo instability, which saturates to an inhomogenous equilibrium with a strong dependence on the magnetic Prandtl number (Pm). Despite its enormously reduced nonlinearity, the dependence of the angular momentum transport on Pm in the quasilinear model is qualitatively similar to that of nonlinear MRI turbulence. This demonstrates the importance of the large-scale dynamo and suggests how dramatically simplified models may be used to gain insight into the astrophysically relevant regimes of very low or high Pm.

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

PubMed Central

Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

2015-01-01

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

PubMed

Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

2016-12-02

Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

Genome-wide mapping of nuclear mitochondrial DNA sequences links DNA replication origins to chromosomal double-strand break formation in Schizosaccharomyces pombe

PubMed Central

Lenglez, Sandrine; Hermand, Damien; Decottignies, Anabelle

2010-01-01

Chromosomal double-strand breaks (DSBs) threaten genome integrity and repair of these lesions is often mutagenic. How and where DSBs are formed is a major question conveniently addressed in simple model organisms like yeast. NUMTs, nuclear DNA sequences of mitochondrial origin, are present in most eukaryotic genomes and probably result from the capture of mitochondrial DNA (mtDNA) fragments into chromosomal breaks. NUMT formation is ongoing and was reported to cause de novo human genetic diseases. Study of NUMTs is likely to contribute to the understanding of naturally occurring chromosomal breaks. We show that Schizosaccharomyces pombe NUMTs are exclusively located in noncoding regions with no preference for gene promoters and, when located into promoters, do not affect gene transcription level. Strikingly, most noncoding regions comprising NUMTs are also associated with a DNA replication origin (ORI). Chromatin immunoprecipitation experiments revealed that chromosomal NUMTs are probably not acting as ORI on their own but that mtDNA insertions occurred directly next to ORIs, suggesting that these loci may be prone to DSB formation. Accordingly, induction of excessive DNA replication origin firing, a phenomenon often associated with human tumor formation, resulted in frequent nucleotide deletion events within ORI3001 subtelomeric chromosomal locus, illustrating a novel aspect of DNA replication-driven genomic instability. How mtDNA is fragmented is another important issue that we addressed by sequencing experimentally induced NUMTs. This highlighted regions of S. pombe mtDNA prone to breaking. Together with an analysis of human NUMTs, we propose that these fragile sites in mtDNA may correspond to replication pause sites. PMID:20688779

In vitro replication of poliovirus

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

Lubinski, J.M.

1986-01-01

Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis ofmore » the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.« less

Regulatory T cells control HIV replication in activated T cells through a cAMP-dependent mechanism

PubMed Central

Moreno-Fernandez, Maria E.; Rueda, Cesar Mauricio; Rusie, Laura K.

2011-01-01

We hypothesized that regulatory T cells (Tregs) could play a beneficial role during HIV infection by controlling HIV replication in conventional T cells (Tcons). Purified Tregs and Tcons from healthy donors were activated separately. Tcons were infected with the X4 or R5 HIV strains and cultured with or without autologous Tregs. Coculture of Tcons and Tregs resulted in a dose-dependent inhibition of Tcon infection, which was significant when a 1:1 Treg:Tcon ratio was used. Treg suppression of HIV infection was largely mediated by contact-dependent mechanisms. Blockage of cytotoxic T-lymphocyte–associated antigen-4 did not significantly reduce Treg function. In contrast, Tregs acted through cAMP-dependent mechanisms, because the decrease of cAMP levels in Tregs, the blockade of gap junction formation between Tregs and Tcons, the blockage of CD39 activity, and the blockage of protein kinase A in Tcons all abolished Treg-mediated suppression of HIV replication. Our data suggest a complex role for Tregs during HIV infection. Although Tregs inhibit specific immune responses, their inhibition of HIV replication in Tcons may play a beneficial role, particularly during early HIV infection, when the effector immune cells are not yet activated. Such a protective role of Tregs could have a profound impact on infection outcome. PMID:21436067

Chk1 promotes replication fork progression by controlling replication initiation

PubMed Central

Petermann, Eva; Woodcock, Mick; Helleday, Thomas

2010-01-01

DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity. PMID:20805465

A new method to calculate the beat-to-beat instability of QT duration in drug-induced long QT in anesthetized dogs.

PubMed

van der Linde, H; Van de Water, A; Loots, W; Van Deuren, B; Lu, H R; Van Ammel, K; Peeters, M; Gallacher, D J

2005-01-01

Instability of QT duration is a marker to predict Torsade de Pointes (TdP) associated with both congenital and drug-induced long QT syndrome. We describe a new method for the quantification of instability of repolarization. Female, adult beagle dogs anesthetized with a potent morphinomimetic were treated with either solvent (n=7) or dofetilide (n=7). Poincaré plots with QT(n) versus QT(n+1) were constructed to visualize the beat-to-beat variation in QT intervals from the lead II ECG. Short-term instability (STI), long-term instability (LTI) and total instability (TI) were quantified by calculating the distances of 30 consecutive data-points from the x and y-coordinate to the "centre of gravity" of the data cluster. Dofetilide at 0.0025 to 0.04 mg/kg i.v. (plasma concentrations of 4+/-0.6 to 41+/-2.7 ng/ml), dose-dependently prolonged QT and QTcV (at 0.04 mg/kg i.v.: QT: 280+/-ms versus 236+/-5 ms with solvent; p<0.05 and QTcV: 290+/-9 ms versus 252+/-4 ms with solvent; p<0.05). Concomitantly, the compound induced an increase in the instability parameters in a similar dose-dependent manner (at 0.04 mg/kg i.v.: TI: 6.8+/-0.9 ms versus 1.7+/-0.3 ms; p<0.05, LTI: 3.6+/-0.5 ms versus 1.0+/-0.2 ms; p<0.05 and STI: 4.2+/-0.6 ms versus 1.0+/-0.2 ms; p<0.05). The increases induced by dofetilide were associated with a high incidence of early afterdepolarizations (EADs) in the endocardial monophasic action potential (in 6 out of the 7 compound-treated animals versus 0 out of the 7 solvent animals; p<0.05). Quantification of beat-to-beat QT instability by our method clearly detects changes in short-term, long-term and total instability induced by dofetilide, already at pre-arrhythmic doses. Dofetilide administration to anesthetized dogs prolongs ventricular repolarization, concomitantly increases beat-to-beat QT instability and induces early after depolarizations (EADs). As such, the use of these parameters in this in vivo model shows clear potential for risk identification in cardiovascular safety assessment.

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

Awe, T. J., E-mail: tjawe@sandia.gov; Jennings, C. A.; McBride, R. D.

Recent experiments at the Sandia National Laboratories Z Facility have, for the first time, studied the implosion dynamics of magnetized liner inertial fusion (MagLIF) style liners that were pre-imposed with a uniform axial magnetic field. As reported [T. J. Awe et al., Phys. Rev. Lett. 111, 235005 (2013)] when premagnetized with a 7 or 10 T axial field, these liners developed 3D-helix-like hydrodynamic instabilities; such instabilities starkly contrast with the azimuthally correlated magneto-Rayleigh-Taylor (MRT) instabilities that have been consistently observed in many earlier non-premagnetized experiments. The helical structure persisted throughout the implosion, even though the azimuthal drive field greatly exceeded themore » expected axial field at the liner's outer wall for all but the earliest stages of the experiment. Whether this modified instability structure has practical importance for magneto-inertial fusion concepts depends primarily on whether the modified instability structure is more stable than standard azimuthally correlated MRT instabilities. In this manuscript, we discuss the evolution of the helix-like instability observed on premagnetized liners. While a first principles explanation of this observation remains elusive, recent 3D simulations suggest that if a small amplitude helical perturbation can be seeded on the liner's outer surface, no further influence from the axial field is required for the instability to grow.« less

Wrinkle surface instability of an inhomogeneous elastic block with graded stiffness

NASA Astrophysics Data System (ADS)

Yang, Shengyou; Chen, Yi-chao

2017-04-01

Surface instabilities have been studied extensively for both homogeneous materials and film/substrate structures but relatively less for materials with continuously varying properties. This paper studies wrinkle surface instability of a graded neo-Hookean block with exponentially varying modulus under plane strain by using the linear bifurcation analysis. We derive the first variation condition for minimizing the potential energy functional and solve the linearized equations of equilibrium to find the necessary conditions for surface instability. It is found that for a homogeneous block or an inhomogeneous block with increasing modulus from the surface, the critical stretch for surface instability is 0.544 (0.456 strain), which is independent of the geometry and the elastic modulus on the surface of the block. This critical stretch coincides with that reported by Biot (1963 Appl. Sci. Res. 12, 168-182. (doi:10.1007/BF03184638)) 53 years ago for the onset of wrinkle instabilities in a half-space of homogeneous neo-Hookean materials. On the other hand, for an inhomogeneous block with decreasing modulus from the surface, the critical stretch for surface instability ranges from 0.544 to 1 (0-0.456 strain), depending on the modulus gradient, and the length and height of the block. This sheds light on the effects of the material inhomogeneity and structural geometry on surface instability.

Elastic instabilities in planar elongational flow of monodisperse polymer solutions

PubMed Central

Haward, Simon J.; McKinley, Gareth H.; Shen, Amy Q.

2016-01-01

We investigate purely elastic flow instabilities in the almost ideal planar stagnation point elongational flow field generated by a microfluidic optimized-shape cross-slot extensional rheometer (OSCER). We use time-resolved flow velocimetry and full-field birefringence microscopy to study the behavior of a series of well-characterized viscoelastic polymer solutions under conditions of low fluid inertia and over a wide range of imposed deformation rates. At low deformation rates the flow is steady and symmetric and appears Newtonian-like, while at high deformation rates we observe the onset of a flow asymmetry resembling the purely elastic instabilities reported in standard-shaped cross-slot devices. However, for intermediate rates, we observe a new type of elastic instability characterized by a lateral displacement and time-dependent motion of the stagnation point. At the onset of this new instability, we evaluate a well-known dimensionless criterion M that predicts the onset of elastic instabilities based on geometric and rheological scaling parameters. The criterion yields maximum values of M which compare well with critical values of M for the onset of elastic instabilities in viscometric torsional flows. We conclude that the same mechanism of tension acting along curved streamlines governs the onset of elastic instabilities in both extensional (irrotational) and torsional (rotational) viscoelastic flows. PMID:27616181

Emotionally Up and Down, Behaviorally to and fro: Drinking Motives Mediate the Synergistic Effects of Urgency and Emotional Instability on Alcohol Outcomes

ERIC Educational Resources Information Center

Dvorak, Robert D.; Kuvaas, Nicholas J.; Lamis, Dorian A.; Pearson, Matthew R.; Stevenson, Brittany L.

2015-01-01

Emotional and behavioral regulation has been linked to coping and enhancement motives and associated with different patterns of alcohol use and problems. The current studies examined emotional instability, urgency, and internal drinking motives as predictors of alcohol dependence symptoms as well as the likelihood and severity of "Diagnostic…

Subcritical saturation of the magnetorotational instability through mean magnetic field generation

NASA Astrophysics Data System (ADS)

Xie, Jin-Han; Julien, Keith; Knobloch, Edgar

2018-03-01

The magnetorotational instability is widely believed to be responsible for outward angular momentum transport in astrophysical accretion discs. The efficiency of this transport depends on the amplitude of this instability in the saturated state. We employ an asymptotic expansion based on an explicit, astrophysically motivated time-scale separation between the orbital period, Alfvén crossing time and viscous or resistive dissipation time-scales, originally proposed by Knobloch and Julien, to formulate a semi-analytical description of the saturated state in an incompressible disc. In our approach a Keplerian shear flow is maintained by the central mass but the instability saturates via the generation of a mean vertical magnetic field. The theory assumes that the time-averaged angular momentum flux and the radial magnetic flux are constant and determines both self-consistently. The results predict that, depending on parameters, steady saturation may be supercritical or subcritical, and in the latter case that the upper (lower) solution branch is always stable (unstable). The angular momentum flux is always outward, consistent with the presence of accretion, and for fixed wavenumber peaks in the subcritical regime. The limit of infinite Reynolds number at large but finite magnetic Reynolds number is also discussed.

A Fuzzy Cognitive Model of aeolian instability across the South Texas Sandsheet

NASA Astrophysics Data System (ADS)

Houser, C.; Bishop, M. P.; Barrineau, C. P.

2014-12-01

Characterization of aeolian systems is complicated by rapidly changing surface-process regimes, spatio-temporal scale dependencies, and subjective interpretation of imagery and spatial data. This paper describes the development and application of analytical reasoning to quantify instability of an aeolian environment using scale-dependent information coupled with conceptual knowledge of process and feedback mechanisms. Specifically, a simple Fuzzy Cognitive Model (FCM) for aeolian landscape instability was developed that represents conceptual knowledge of key biophysical processes and feedbacks. Model inputs include satellite-derived surface biophysical and geomorphometric parameters. FCMs are a knowledge-based Artificial Intelligence (AI) technique that merges fuzzy logic and neural computing in which knowledge or concepts are structured as a web of relationships that is similar to both human reasoning and the human decision-making process. Given simple process-form relationships, the analytical reasoning model is able to map the influence of land management practices and the geomorphology of the inherited surface on aeolian instability within the South Texas Sandsheet. Results suggest that FCMs can be used to formalize process-form relationships and information integration analogous to human cognition with future iterations accounting for the spatial interactions and temporal lags across the sand sheets.

Effects of stochastic field lines on the pressure driven MHD instabilities in the Large Helical Device

NASA Astrophysics Data System (ADS)

Ohdachi, Satoshi; Watanabe, Kiyomasa; Sakakibara, Satoru; Suzuki, Yasuhiro; Tsuchiya, Hayato; Ming, Tingfeng; Du, Xiaodi; LHD Expriment Group Team

2014-10-01

In the Large Helical Device (LHD), the plasma is surrounded by the so-called magnetic stochastic region, where the Kolmogorov length of the magnetic field lines is very short, from several tens of meters and to thousands meters. Finite pressure gradient are formed in this region and MHD instabilities localized in this region is observed since the edge region of the LHD is always unstable against the pressure driven mode. Therefore, the saturation level of the instabilities is the key issue in order to evaluate the risk of this kind of MHD instabilities. The saturation level depends on the pressure gradient and on the magnetic Reynolds number; there results are similar to the MHD mode in the closed magnetic surface region. The saturation level in the stochastic region is affected also by the stocasticity itself. Parameter dependence of the saturation level of the MHD activities in the region is discussed in detail. It is supported by NIFS budget code ULPP021, 028 and is also partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research 26249144, by the JSPS-NRF-NSFC A3 Foresight Program NSFC: No. 11261140328.

Interplay between electric fields generated by reconnection and by secondary processes

NASA Astrophysics Data System (ADS)

Lapenta, G.; Innocenti, M. E.; Pucci, F.; Cazzola, E.; Berchem, J.; Newman, D. L.; El-Alaoui, M.; Walker, R. J.; Goldman, M. V.; Ergun, R.

2017-12-01

Reconnection regions are surrounded by several sources of free energy that push reconnection towards a turbulent regime: beams can drive streaming instabilities, currents can drive tearing like secondary instabilities, velocity and density shears can drive Kelvin-Helmholtz or Rayleigh-Taylor type of instabilities. The interaction between these instabilities can be very complex. For instance, from a kinetic point of view, instabilities resulting from shears are intermixed with drift-type instabilities, such as drift-kink, kink driven by relative species drift, lower hybrid modes of the electrostatic or electromagnetic type. In addition, the interaction with reconnection is two ways: reconnection causes the conditions for those instabilities to develop while the instabilities alter the progress of reconnection. Although MMS has observed features that can be associated with such instabilities: strong localized parallel electric fields (monopolar and bipolar), fluctuations in the drift range (lower hybrid, whistler), it has been difficult to determine which ones operate and how they differ depending on the symmetric and asymmetric reconnection configurations observed in the magnetotail and at the magnetopause, respectively. We present a comparison between the results of kinetic simulations obtained for typical magnetotail and the magnetopause configurations, using for each of them both analytical equilibria and results of global MHD simulations to initialize the iPIC3D simulations. By selecting what drivers (e.g. shear/no shear) are present, we can identify what instabilities develop and determine their effects on the progression of reconnection in the magnetotail and at the magnetopause. We focus especially on the role of drift waves and whistler instabilities, and discuss our results by comparing them with MMS observations.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

PubMed Central

Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

2016-01-01

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

The simulation of the geosynchronous Earth orbit plasma environment in Chamber A: An assessment of possible experimental investigations

NASA Technical Reports Server (NTRS)

Bernstein, W.

1981-01-01

The possible use of Chamber A for the replication or simulation of space plasma physics processes which occur in the geosynchronous Earth orbit (GEO) environment is considered. It is shown that replication is not possible and that scaling of the environmental conditions is required for study of the important instability processes. Rules for such experimental scaling are given. At the present time, it does not appear technologically feasible to satisfy these requirements in Chamber A. It is, however, possible to study and qualitatively evaluate the problem of vehicle charging at GEO. In particular, Chamber A is sufficiently large that a complete operational spacecraft could be irradiated by beams and charged to high potentials. Such testing would contribute to the assessment of the operational malfunctions expected at GEO and their possible correction. However, because of the many tabulated limitations in such a testing programs, its direct relevance to conditions expected in the geo environment remains questionable.

Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control.

PubMed

Gallina, Irene; Colding, Camilla; Henriksen, Peter; Beli, Petra; Nakamura, Kyosuke; Offman, Judith; Mathiasen, David P; Silva, Sonia; Hoffmann, Eva; Groth, Anja; Choudhary, Chunaram; Lisby, Michael

2015-03-30

DNA replication stress is a source of genomic instability. Here we identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1--together with Mrc1/Claspin, Pph3, the chaperonin containing TCP1 (CCT) and 25 other proteins--define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to Cmr1, its human orthologue WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that Cmr1/WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins.

Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control

PubMed Central

Gallina, Irene; Colding, Camilla; Henriksen, Peter; Beli, Petra; Nakamura, Kyosuke; Offman, Judith; Mathiasen, David P.; Silva, Sonia; Hoffmann, Eva; Groth, Anja; Choudhary, Chunaram; Lisby, Michael

2015-01-01

DNA replication stress is a source of genomic instability. Here we identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1—together with Mrc1/Claspin, Pph3, the chaperonin containing TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to Cmr1, its human orthologue WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that Cmr1/WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins. PMID:25817432

ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress.

PubMed

Liu, Rui; King, Ashleigh; Hoch, Nicolas C; Chang, Catherine; Kelly, Gemma L; Deans, Andrew J; Heierhorst, Jörg

2017-09-01

The ATM kinase plays critical roles in the response to DNA double-strand breaks, and can also be activated by prolonged DNA replication blocks. It has recently been proposed that replication stress-dependent ATM activation is mediated by ASCIZ (also known as ATMIN, ZNF822), an essential developmental transcription factor. In contrast, we show here that ATM activation, and phosphorylation of its substrates KAP1, p53 and H2AX in response to the replication blocking agent aphidicolin was unaffected in both immortalized and primary ASCIZ/ATMIN-deficient murine embryonic fibroblasts compared to control cells. Similar results were also obtained in human ASCIZ/ATMIN-deleted lymphoma cells. The results demonstrate that ASCIZ/ATMIN is dispensable for ATM activation, and contradict the previously reported dependence of ATM on ASCIZ/ATMIN. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Collective Effects in a Diffraction Limited Storage Ring

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

Nagaoka, Ryutaro; Bane, Karl L.F.

Our paper gives an overview of collective effects that are likely to appear and possibly limit the performance in a diffraction-limited storage ring (DLSR) that stores a high-intensity ultra-low-emittance beam. Beam instabilities and other intensity-dependent effects that may significantly impact the machine performance are covered. The latter include beam-induced machine heating, Touschek scattering, intra-beam scattering, as well as incoherent tune shifts. The general trend that the efforts to achieve ultra-low emittance result in increasing the machine coupling impedance and the beam sensitivity to instability is reviewed. The nature of coupling impedance in a DLSR is described, followed by a seriesmore » of potentially dangerous beam instabilities driven by the former, such as resistive-wall, TMCI (transverse mode coupling instability), head-tail and microwave instabilities. Additionally, beam-ion and CSR (coherent synchrotron radiation) instabilities are also treated. Means to fight against collective effects such as lengthening of the bunch with passive harmonic cavities and bunch-by-bunch transverse feedback are introduced. Numerical codes developed and used to evaluate the machine coupling impedance, as well as to simulate beam instability using the former as inputs are described.« less

Collective Effects in a Diffraction Limited Storage Ring

DOE PAGES

Nagaoka, Ryutaro; Bane, Karl L.F.

2015-10-20

Our paper gives an overview of collective effects that are likely to appear and possibly limit the performance in a diffraction-limited storage ring (DLSR) that stores a high-intensity ultra-low-emittance beam. Beam instabilities and other intensity-dependent effects that may significantly impact the machine performance are covered. The latter include beam-induced machine heating, Touschek scattering, intra-beam scattering, as well as incoherent tune shifts. The general trend that the efforts to achieve ultra-low emittance result in increasing the machine coupling impedance and the beam sensitivity to instability is reviewed. The nature of coupling impedance in a DLSR is described, followed by a seriesmore » of potentially dangerous beam instabilities driven by the former, such as resistive-wall, TMCI (transverse mode coupling instability), head-tail and microwave instabilities. Additionally, beam-ion and CSR (coherent synchrotron radiation) instabilities are also treated. Means to fight against collective effects such as lengthening of the bunch with passive harmonic cavities and bunch-by-bunch transverse feedback are introduced. Numerical codes developed and used to evaluate the machine coupling impedance, as well as to simulate beam instability using the former as inputs are described.« less

Bet-hedging as a complex interaction among developmental instability, environmental heterogeneity, dispersal, and life-history strategy.

PubMed

Scheiner, Samuel M

2014-02-01

One potential evolutionary response to environmental heterogeneity is the production of randomly variable offspring through developmental instability, a type of bet-hedging. I used an individual-based, genetically explicit model to examine the evolution of developmental instability. The model considered both temporal and spatial heterogeneity alone and in combination, the effect of migration pattern (stepping stone vs. island), and life-history strategy. I confirmed that temporal heterogeneity alone requires a threshold amount of variation to select for a substantial amount of developmental instability. For spatial heterogeneity only, the response to selection on developmental instability depended on the life-history strategy and the form and pattern of dispersal with the greatest response for island migration when selection occurred before dispersal. Both spatial and temporal variation alone select for similar amounts of instability, but in combination resulted in substantially more instability than either alone. Local adaptation traded off against bet-hedging, but not in a simple linear fashion. I found higher-order interactions between life-history patterns, dispersal rates, dispersal patterns, and environmental heterogeneity that are not explainable by simple intuition. We need additional modeling efforts to understand these interactions and empirical tests that explicitly account for all of these factors.

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

PubMed

Chappell, William H; Gautam, Dipendra; Ok, Suzan T; Johnson, Bryan A; Anacker, Daniel C; Moody, Cary A

2015-12-23

High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A hydrodynamic instability for rip currents

NASA Astrophysics Data System (ADS)

Yu, J.; Chen, S.

2012-12-01

In Yu (2006), it is shown that on a planar beach a hydrodynamic instability can occur, leading to the generation of circulations that are related to rip currents. This instability depends on the mutual interaction of waves and evolving currents. In this study, we extend the analysis to non-planar profiles, include barred beaches. Both propagating and non-propagating unstable solutions are found. The oscillatory nature (in space and in time) of the propagating solutions may provide one explanation of the episodic events of rip currents on some beaches. Support from National Science Foundation (Grant CBET-0845957) is acknowledged.

Radiative Instabilities in Three-Dimensional Astrophysical Masers

NASA Technical Reports Server (NTRS)

Scappaticci, Gerardo A.; Watson, William D.

1995-01-01

Inherent instabilities in the radiative transfer for astrophysical masers have been recognized and calculated in the linear maser idealization in our previous investigations. The same instabilities are now shown to occur in the more realistic, three-dimensional geometries. Fluctuations in the emergent flux result and may be related to the observed fluctuations in the radiative flux from the 1665 MHz OH masers that have been reported to occur on timescales as short as 1000 s. The time-dependent differential equations of radiative transfer are solved numerically for three-dimensional astrophysical masers. Computations are performed for spherical and elongated (rectangular parallelepiped) geometries.

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

PubMed

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

2015-09-01

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

Twirling and Whirling: Viscous Dynamics of Rotating Elastica

NASA Astrophysics Data System (ADS)

Powers, Thomas R.; Wolgemuth, Charles W.; Goldstein, Raymond E.

1999-11-01

Motivated by diverse phenomena in cellular biophysics, including bacterial flagellar motion and DNA transcription and replication, we study the overdamped nonlinear dynamics of a rotationally forced filament with twist and bend elasticity. The competition between twist diffusion and writhing instabilities is described by a novel pair of coupled PDEs for twist and bend evolution. Analytical and numerical methods elucidate the twist-bend coupling and reveal two dynamical regimes separated by a Hopf bifurcation: (i) diffusion-dominated axial rotation, or twirling, and (ii) steady-state crankshafting motion, or whirling. The consequences of these phenomena for self-propulsion are investigated, and experimental tests proposed.

Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells

PubMed Central

Zhou, Yunying; Zhang, Qishu; Gao, Ge; Zhang, Xiaoli; Liu, Yafei; Yuan, Shoudao

2016-01-01

ABSTRACT The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our studies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. IMPORTANCE The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated in E7-expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. PMID:27099318

Frequencies of Flare Occurrence: Interaction between Convection and Coronal Loops

NASA Astrophysics Data System (ADS)

Mullan, D. J.; Paudel, R. R.

2018-02-01

Observations of solar and stellar flares have revealed the presence of power-law dependences between the flare energy and the time interval between flares. Various models have been proposed to explain these dependences and the numerical value of the power-law indices. Here, we propose a model in which convective flows in granules force the footpoints of coronal magnetic loops, which are frozen-in to photospheric gas, to undergo a random walk. In certain conditions, this can lead to a twist in the loop, which drives the loop unstable if the twist exceeds a critical value. The possibility that a solar flare is caused by such a twist-induced instability in a loop has been in the literature for decades. Here, we quantify the process in an approximate way with a view to replicating the power-law index. We find that, for relatively small flares, the random walk twisting model leads to a rather steep power-law slope that agrees very well with the index derived from a sample of 56,000+ solar X-ray flares reported by the GOES satellites. For relatively large flares, we find that the slope of the power law is shallower. The empirical power-law slopes reported for flare stars also have a range that overlaps with the slopes obtained here. We suggest that in the coolest stars, a significant change in slope should occur when the frozen-flux assumption breaks down due to low electrical conductivity.

Chromosome dynamics in the yeast interphase nucleus.

PubMed

Heun, P; Laroche, T; Shimada, K; Furrer, P; Gasser, S M

2001-12-07

Little is known about the dynamics of chromosomes in interphase nuclei. By tagging four chromosomal regions with a green fluorescent protein fusion to lac repressor, we monitored the movement and subnuclear position of specific sites in the yeast genome, sampling at short time intervals. We found that early and late origins of replication are highly mobile in G1 phase, frequently moving at or faster than 0.5 micrometers/10 seconds, in an energy-dependent fashion. The rapid diffusive movement of chromatin detected in G1 becomes constrained in S phase through a mechanism dependent on active DNA replication. In contrast, telomeres and centromeres provide replication-independent constraint on chromatin movement in both G1 and S phases.

The Dot/Icm effector SdhA is necessary for virulence of Legionella pneumophila in Galleria mellonella and A/J mice.

PubMed

Harding, Clare R; Stoneham, Charlotte A; Schuelein, Ralf; Newton, Hayley; Oates, Clare V; Hartland, Elizabeth L; Schroeder, Gunnar N; Frankel, Gad

2013-07-01

Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ΔsdhA mutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage.

PubMed Central

Early, Anne; Drury, Lucy S; Diffley, John F X

2004-01-01

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start. PMID:15065654

A novel subviral agent associated with a geminivirus: The first report of a DNA satellite

PubMed Central

Dry, Ian B.; Krake, Leslie R.; Rigden, Justin E.; Rezaian, M. Ali

1997-01-01

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem–loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication. PMID:9192696

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

PubMed

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

2016-07-26

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

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

PubMed Central

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

Simple systems that exhibit self-directed replication

NASA Technical Reports Server (NTRS)

Reggia, James A.; Armentrout, Steven L.; Chou, Hui-Hsien; Peng, Yun

1993-01-01

Biological experience and intuition suggest that self-replication is an inherently complex phenomenon, and early cellular automata models support that conception. More recently, simpler computational models of self-directed replication called sheathed loops have been developed. It is shown here that 'unsheathing' these structures and altering certain assumptions about the symmetry of their components leads to a family of nontrivial self-replicating structures some substantially smaller and simpler than those previously reported. The dependence of replication time and transition function complexity on initial structure size, cell state symmetry, and neighborhood are examined. These results support the view that self-replication is not an inherently complex phenomenon but rather an emergent property arising from local interactions in systems that can be much simpler than is generally believed.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

PubMed

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Parametric instability analysis of truncated conical shells using the Haar wavelet method

NASA Astrophysics Data System (ADS)

Dai, Qiyi; Cao, Qingjie

2018-05-01

In this paper, the Haar wavelet method is employed to analyze the parametric instability of truncated conical shells under static and time dependent periodic axial loads. The present work is based on the Love first-approximation theory for classical thin shells. The displacement field is expressed as the Haar wavelet series in the axial direction and trigonometric functions in the circumferential direction. Then the partial differential equations are reduced into a system of coupled Mathieu-type ordinary differential equations describing dynamic instability behavior of the shell. Using Bolotin's method, the first-order and second-order approximations of principal instability regions are determined. The correctness of present method is examined by comparing the results with those in the literature and very good agreement is observed. The difference between the first-order and second-order approximations of principal instability regions for tensile and compressive loads is also investigated. Finally, numerical results are presented to bring out the influences of various parameters like static load factors, boundary conditions and shell geometrical characteristics on the domains of parametric instability of conical shells.

The Multivariate Roles of Family Instability and Interparental Conflict in Predicting Children’s Representations of Insecurity in the Family System and Early School Adjustment Problems

PubMed Central

Coe, Jesse L.; Davies, Patrick T.; Sturge-Apple, Melissa L.

2016-01-01

This study examined the moderating role of family instability in relations involving destructive interparental conflict, children’s internal representations of insecurity in the family system, and their early school maladjustment. Two hundred forty-three preschool children (M age = 4.60 years; 56% girls) and their families participated in this multi-method (i.e., observations, structured interview, surveys) multi-informant (i.e., observer, parent, teacher), longitudinal study. Findings indicated that the mediational role of children’s insecure family representations in the pathway between destructive interparental conflict and children’s adjustment problems varied significantly depending on the level of family instability. Interparental conflict was specifically associated with insecure family representations only under conditions of low family instability. In supporting the role of family instability as a vulnerable-stable risk factor, follow up analyses revealed that children’s concerns about security in the family were uniformly high under conditions of heightened instability regardless of their level of exposure to interparental conflict. PMID:27146062

Marangoni instability in a thin film heated from below: Effect of nonmonotonic dependence of surface tension on temperature

NASA Astrophysics Data System (ADS)

Sarma, Rajkumar; Mondal, Pranab Kumar

2018-04-01

We investigate Marangoni instability in a thin liquid film resting on a substrate of low thermal conductivity and separated from the surrounding gas phase by a deformable free surface. Considering a nonmonotonic variation of surface tension with temperature, here we analytically derive the neutral stability curve for the monotonic and oscillatory modes of instability (for both the long-wave and short-wave perturbations) under the framework of linear stability analysis. For the long-wave instability, we derive a set of amplitude equations using the scaling k ˜(Bi) 1 /2 , where k is the wave number and Bi is the Biot number. Through this investigation, we demonstrate that for such a fluid layer upon heating from below, both monotonic and oscillatory instability can appear for a certain range of the dimensionless parameters, viz., Biot number (Bi ) , Galileo number (Ga ) , and inverse capillary number (Σ ) . Moreover, we unveil, through this study, the influential role of the above-mentioned parameters on the stability of the system and identify the critical values of these parameters above which instability initiates in the liquid layer.

Asymptotic Laws of Thermovibrational Convecton in a Horizontal Fluid Layer

NASA Astrophysics Data System (ADS)

Smorodin, B. L.; Myznikova, B. I.; Keller, I. O.

2017-02-01

Theoretical study of convective instability is applied to a horizontal layer of incompressible single-component fluid subjected to the uniform steady gravity, longitudinal vibrations of arbitrary frequency and initial temperature difference. The mathematical model of thermovibrational convection has the form of initial boundary value problem for the Oberbeck-Boussinesq system of equations. The problems are solved using different simulation strategies, like the method of averaging, method of multiple scales, Galerkin approach, Wentzel-Kramers-Brillouin method and Floquet technique. The numerical analysis has shown that the effect of vibrations on the stability threshold is complex: vibrations can either stabilize or destabilize the basic state depending on values of the parameters. The influence of the Prandtl number on the instability thresholds is investigated. The asymptotic behaviour of critical values of the parameters is studied in two limiting cases: (i) small amplitude and (ii) low frequency of vibration. In case (i), the instability is due to the influence of thermovibrational mechanism on the classical Rayleigh-Benard convective instability. In case (ii), the nature of the instability is related to the instability of oscillating counter-streams with a cubic profile.

Local parametric instability near elliptic points in vortex flows under shear deformation

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

Koshel, Konstantin V., E-mail: kvkoshel@poi.dvo.ru; Institute of Applied Mathematics, FEB RAS, 7, Radio Street, Vladivostok 690022; Far Eastern Federal University, 8, Sukhanova Street, Vladivostok 690950

The dynamics of two point vortices embedded in an oscillatory external flow consisted of shear and rotational components is addressed. The region associated with steady-state elliptic points of the vortex motion is established to experience local parametric instability. The instability forces the point vortices with initial positions corresponding to the steady-state elliptic points to move in spiral-like divergent trajectories. This divergent motion continues until the nonlinear effects suppress their motion near the region associated with the steady-state separatrices. The local parametric instability is then demonstrated not to contribute considerably to enhancing the size of the chaotic motion regions. Instead, themore » size of the chaotic motion region mostly depends on overlaps of the nonlinear resonances emerging in the perturbed system.« less

Suppression of the Saffman-Taylor instability through injection of a finite slug of polymer

NASA Astrophysics Data System (ADS)

Beeson-Jones, Timothy H.; Woods, Andrew W.

2014-11-01

During secondary oil recovery, relatively mobile water can channel through oil owing to the Saffman-Taylor instability. Injection of a finite slug of polymer solution from a central well prior to the water flood suppresses the growth of the instability by reducing the adverse mobility ratio at the leading interface. A linear stability analysis of an axisymmetric base state identifies how perturbations on the leading and trailing interfaces become coupled. It also reveals the dependence of the long-time algebraic growth of each mode on the mobility ratios across the two interfaces. The viscosity of the polymer solution which minimizes the growth rate of the instability is identified, and the impact of different slug sizes on this growth is described. Funded by EPSRC & BP.

Spatial aspects of prebiotic replicator coexistence and community stability in a surface-bound RNA world model

PubMed Central

2013-01-01

Background The coexistence of macromolecular replicators and thus the stability of presumed prebiotic replicator communities have been shown to critically depend on spatially constrained catalytic cooperation among RNA-like modular replicators. The necessary spatial constraints might have been supplied by mineral surfaces initially, preceding the more effective compartmentalization in membrane vesicles which must have been a later development of chemical evolution. Results Using our surface-bound RNA world model – the Metabolic Replicator Model (MRM) platform – we show that the mobilities on the mineral substrate surface of both the macromolecular replicators and the small molecules of metabolites they produce catalytically are the key factors determining the stable persistence of an evolvable metabolic replicator community. Conclusion The effects of replicator mobility and metabolite diffusion on different aspects of replicator coexistence in MRM are determined, including the maximum attainable size of the metabolic replicator system and its resistance to the invasion of parasitic replicators. We suggest a chemically plausible hypothetical scenario for the evolution of the first protocell starting from the surface-bound MRM system. PMID:24053177