MECHANISMS PRESERVING GENOME INTEGRITY IN SACCHAROMYCES CEREVISIAE

The integrity of the genome is continuously jeopardized by endogenous reactive byproducts of cellular metabolism and genotoxic insults by environmental agents, as well as by the DNA transactions (replication, transcription and recombination) required for cell survival and proliferation. Failure of the mechanisms deputed to the maintenance of genome integrity leads to genome instability, which is a hallmark of cancer and a driving force of tumorigenesis. To fully understand the mechanisms leading to genome instability and the cellular pathways counteracting them, three basic tasks must be achieved: i) identify all the genes implicated in the control of genome integrity; ii) unravel their biological role; iii) define the mechanistic molecular details of the processes in which they are implicated. This thesis describes work performed in the budding yeast Saccharomyces cerevisiae to explore the genome stability landscape at all these three levels. This model system is extremely useful for two main reasons: a) its high genetic tractability allows the application of genome-wide genetic screenings; b) the large conservation of the genome integrity pathways allows to extend the findings obtained in yeast to other eukaryotic organisms. We performed a genome-wide screen, based on the overexpression of the DDC2 DNA damage checkpoint gene in the yeast deletion collection, to identify genome stability genes on the basis of spontaneous accumulation of endogenous DNA damage in the corresponding mutant strains. Our screen identified several genes implicated in the control of genome integrity, highlighting, in particular, a key role for pathways protecting against oxidative stress. We present here the preliminary characterization of a new genome integrity gene, VID22. We also investigated the mechanisms counteracting a newly discovered source of genome instability, namely ribonucleotides (rNTPs) incorporated in genomic DNA during replication. We uncovered a role for RNase H enzymes, template switch pathways and Pol \u3b6 translesion polymerase in protecting from misincorporated rNTPs. Given that mutations in any of the three human RNase H2 subunits were proven to cause Aicardi-Gouti\ue9res Syndrome, these results might contribute to shed light on the complex and largely unknown pathogenetic mechanism of this rare genetic disease. Finally, we studied the molecular details underlying the role of Rad9 mediator protein in DNA damage checkpoint activation, exploring the dynamics of Rad9 dimerization, chromatin binding, CDK-dependent phosphorylation and checkpoint activation in G1 and M phases of the cell cycle; in particular, we characterized an M-phase specific pathway for checkpoint activation which is relies on Rad9-Dpb11 interaction

Pengendalian Gulma pada Kebun Kelapa Sawit (Elaeis Guineensis Jacq)k2idan Kebunmasyarakat di Desa Bangko Kiri Kecamatanbangko Pusako Kabupaten Rokan Hilirprovinsi Riau

The research purpose tounderstandingthe weedcontrol anddominant weeds of palm oil (Elaeis guineensis Jacq.) plantation. The research was carried out in the village of Bangko Pusako Kabupaten Rokan Hilir from May until July 2014 with method survey, analyzed by quantitative statistics and were present in deskriptive.The sample collection by purposively sampling based on land area and age of plant. The total sample taken were 42 samples or 10 percent of 416 farmers palm oil and for the type of dominant weed done in vegetation with analysis quadrantmethod.Parameters that observed wereorigin of seed, cropping pattern distance, distance cropping, the population of plants , weeds dominant type , weed management techniques, rotation management, kind of a herbicide and dose of herbicide.The research results show farmers sample used weeds management technique with chemically and mechanically 76,2 % rate and 47.7 % farmers usedrotation management for 6 times a year, 52,38 % farmers used herbicide with active compoud paraquat-dimethylamine, 52,3 % farmers used3l / hadose while K2I farm also do the same thing, but the management carry out twice a year. The weeds dominant type in K2I and people farmisSpotaneum saccharum L. (shaken) 56,25 %

The Grizzly, March 7, 1986

Meyer to Undergo Surgery • Teachers Always Looking to the Future: NASA Finalists Speak to Some U.C. Students • Garbage: It\u27s Expensive Stuff • Letters: CAB Responds to Only at Ursinus Comments; Student Offended by Walkman Listener at Haydn Concert • Bomberger Concerts Deserve Crowd, Too • Tie for First in Air Band • How to get that \u27A\u27 • It\u27s All in Good Fun Guys • What are You Doing Next Week? • Nuclear War as a Just War • Women\u27s Studies Offered in Fall • Mer Chicks a Success at MAC\u27s • U.C. Boys Bearing Down • Women\u27s Lacrosse Preview • Coach Brown Named Ass\u27t Athletic Director • Townshend Strikes Gold With White City LP • Tolkien Collection On Display in Myrin • Faculty Views of Pledginghttps://digitalcommons.ursinus.edu/grizzlynews/1160/thumbnail.jp

VID22 counteracts G-quadruplex-induced genome instability

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism

RNAi Screening Implicates a SKN-1-Dependent Transcriptional Response in Stress Resistance and Longevity Deriving from Translation Inhibition

Caenorhabditis elegans SKN-1 (ortholog of mammalian Nrf1/2/3) is critical for oxidative stress resistance and promotes longevity under reduced insulin/IGF-1-like signaling (IIS), dietary restriction (DR), and normal conditions. SKN-1 inducibly activates genes involved in detoxification, protein homeostasis, and other functions in response to stress. Here we used genome-scale RNA interference (RNAi) screening to identify mechanisms that prevent inappropriate SKN-1 target gene expression under non-stressed conditions. We identified 41 genes for which knockdown leads to activation of a SKN-1 target gene (gcs-1) through skn-1-dependent or other mechanisms. These genes correspond to multiple cellular processes, including mRNA translation. Inhibition of translation is known to increase longevity and stress resistance and may be important for DR-induced lifespan extension. One model postulates that these effects derive from reduced energy needs, but various observations suggest that specific longevity pathways are involved. Here we show that translation initiation factor RNAi robustly induces SKN-1 target gene transcription and confers skn-1-dependent oxidative stress resistance. The accompanying increases in longevity are mediated largely through the activities of SKN-1 and the transcription factor DAF-16 (FOXO), which is required for longevity that derives from reduced IIS. Our results indicate that the SKN-1 detoxification gene network monitors various metabolic and regulatory processes. Interference with one of these processes, translation initiation, leads to a transcriptional response whereby SKN-1 promotes stress resistance and functions together with DAF-16 to extend lifespan. This stress response may be beneficial for coping with situations that are associated with reduced protein synthesis

Fluid-Structure Interaction and Co-Simulation: Analysis of a Beam-Supported Sphere for VIV Application

The recent developments in numerical tools and computing resources seem to provide a suitable environment to perform numerical analyses of Fluid-Structure Interaction problems. The Co-Simulation technique, in particular, develops the idea of coupling a CFD software with a structural one in order to simulate complex FSI phenomena with a partitioned approach, stressing the concept of software modularity. In this way, it is possible to adopt software tools at the cutting edge of technology. Nonetheless, several difficulties may arise in the choice of the partitioning scheme and of the algorithmic details for the step-by-step time integration. This paper deals with the application of the Co-Simulation technique to a benchmark case experimentally investigated in previous works: the vortex-induced vibrations (VIV) of a beam supported sphere (that is, a sphere fixed to the end of a slender cantilever beam) in a free surface flow. This problem is challenging although apparently simple and it seems quite absent from literature so far. In this paper, the computational issues are thoroughly investigated and the model is validated by comparison with the experimental data. In this way, a robust framework is created in order to deal with VIV problems

RNaseH2 plays a role in the maintenance of genome stability

Ribonuclease H (RNaseH) is a family of evolutionally conserved enzymes capable of cleaving the RNA moiety in RNA:DNA hybrid molecules. Eukaryotic cells are provided with RNaseHI and RnaseH2 activities. The main differences between these two enzymes rest in substrate specificity. In fact only RNaseH2 is capable to recognize and cleave a single ribonucleotide in a duplex DNA molecule. Mutations in the RNaseH2 enzyme, are found in a subset of patients suffering of a rare genetic disease, called Aicardi-Gouti\ue8res Syndrome (AGS). RNA:DNA hybrids are transient intermediates occurring during several biological processes, such as DNA replication, telomere metabolism, retrovirus replication, retroelements mobilization and transcription (i.e. R-loops). These hybrid molecules are physiologically short-lived, but in some conditions they can generate pathological structures that need to be tightly controlled in order to avoid an increase in genomic instability that is known to be linked to cancer predisposition. We are studying the role of RNaseH in controlling genome integrity in normal and replication stress conditions, using S.cerevisiae cells as a model system. Our results indicate that yeast RNaseH mutant cells are sensitive to genotoxic treatments. We have characterized the biological process responsible for this phenotype and we have identified the molecular pathways that allows survival of the RNaseH mutants in the presence of replication stress

Systematic approaches for the definition of the DNA damage response pathways in budding yeast

Upon DNA damage cells activate a genetically controlled pathway, known as DNA damage checkpoint, which is devoted to helping cells cope with genomic lesions. Much of the molecular details underlying this response are now understood, but the integration between checkpoint factors and other cellular pathways are still mostly undefined. We are trying to identify new mechanisms involved in the maintenance of genome stability, using multiple parallel approaches in the genetically tractable model system Saccharomyces cerevisiae. 1. Identification of the substrates of checkpoint kinases. Taking advantage of a biotin ligase module, derived from E.coli, we generated a yeast strain which expresses a Rad53 protein, which contains also a biotin ligase activity. This chimeric protein is fully active in the checkpoint response and in the preservation of stalled replication forks. Diploid cells obtained by crossing this strain with a collection of yeast cells expressing individual genes tagged with a biotinilation motif, will be screened for targets of the Rad53 checkpoint kinase. 2. Identification of new genes required for the maintenance of genome integrity. Several mutations in genes known to be involved in the preservation of genome stability cause a variable level of phosphorylation of Rad53 in physiological growth conditions. This is likely due to a higher background level of endogenous genomic instability. Overexpression of yeast checkpoint proteins in such strains leads to cell lethality, probably due to the imposition of cell cycle arrest caused by the simultaneous presence of the mutation and a hypersensitive checkpoint pathway. We will take advantage of this strategy to perform an automated systematic search of a yeast gene deletion collection for mutants which are synthetically lethal when the checkpoint is overstimulated. 3. Systematic identification of new factors interacting with checkpoint factors. We are generating a yeast strain collection expressing checkpoint proteins as a two-hybrid bait. We also produced a yeast prey library which is expressed in cells of the opposite mating type. By large scale mating experiments and taking advantage of a fluorescent automated screening methodology, we will systematically search for yeast proteins physically interacting with all the factors involved in the checkpoint signal transduction cascade