1,720,982 research outputs found
Functional Analysis of the Cell Cycle Protein Dpb11 in Response to DNA Damage and Replicative Stress
Full text linkDNA molecule is complex, fragile and can suffer different damages. Specific DNA repair mechanisms were evolved to respond to these challenges, and to allow a faithful transmission of genetic information throughout generations. If the damaging conditions are extensive, a mechanism called DNA damage checkpoint takes care of arresting the progression of the cell division cycle to allow the cell to repair the damage before proceeding further. Genes involved in the DNA damage checkpoint are conserved throughout evolution and mutations in the human genes are known to produce severe illnesses - like Ataxia Telangiectasia - and genomic instability, which is usually considered as the onset of cancer: indeed checkpoint genes, like BRCA1, were found to be mutated in different types of cancers.
The yeast Saccharomyces cerevisiae has been widely used to study the DNA damage checkpoint because, despite its evolutionary distance, the easiness in generating knockout and mutant strains has facilitated the understanding of the underlying mechanisms. In this yeast, as in humans, the signal that activates the checkpoint is represented by the ssDNA covered by RPA, to which many different checkpoint and repair factors are recruited. ssDNA signals are responsible for the activation of Mec1 (hATR), the apical kinase of the checkpoint pathway, but in humans two other factors are required for this signalling to occur: a ring-like heterotrimer - the PCNA-like complex - which is loaded onto DNA in response to damage and which recruits the second factor, TopBP1. Once active, Mec1 kinase phosphorylates a series of substrates, among which there is the Ddc1 subunit of the PCNA-like complex, and the Rad9 protein; phosphorylated Rad9 allows the recruitment of Rad53, the central kinase of the checkpoint whose Mec1-dependent activation contributes to cell survival after DNA damage and replication stress. To be phosphorylated by DNA-bound Mec1, the Rad9 protein must be recruited to chromatin: this process involves the binding of a Rad9 domain - the Tudor domain - to a methylated lysine on histone H3. Indeed, cells mutated in the conserved H3 lysine, in the Tudor domain or in the histone methyltransferase Dot1 are defective in Rad9 and Rad53 phosphorylation when DNA is damaged in the G1 phase of the cell cycle. Surprisingly, when these mutants receive a DNA damage in mitosis, they are still able to phosphorylate Rad9 and Rad53, suggesting the presence of a second pathway that, in M phase, provides an alternative way for Rad9 to be phosphorylated.
In this thesis evidences regarding this alternative pathway for Rad9 recruitment and phosphorylation are provided. This pathway depends upon the C-terminal tail of Dpb11, the yeast homologue of human TopBP1, and on the Mec1-dependent phosphorylation of threonine 602 of the Ddc1 subunit of the PCNA-like complex. We show that Dpb11 itself is phosphorylated after DNA damage and that this phosphorylation is reduced in the presence of a non-phosphorylatable 602-residue on Ddc1, suggesting that in these conditions Dpb11 cannot be functionally recruited. Supporting this idea the two-hybrid interaction between Ddc1 and Dpb11 requires the presence of a functional Mec1 kinase.
Although being capable of in vitro stimulation of Mec1 kinase activity, after UV irradiation in M phase, Dpb11 is not required for Mec1 to phosphorylate its binding partner Ddc2. On the other hand, we provide evidences that Dpb11 performs its Mec1 activation task during the response to global replication stress; indeed Dpb11 and the PCNA-like complex are independently required to obtain a proper phosphorylation of histone H2A - here used as a marker of Mec1 kinase activity - and a full Rad53 activation. Consistent with this observation ddc1Δdpb11-1 mutants are extremely sensitive to chronic exposition to hydroxyurea, a commonly used chemotherapeutic drug that generates replication stress by reducing the concentration of dNTPs in the cell. We also provide evidence that this lethality is not due to classical checkpoint functions like the stabilisation of stalled replication forks or the ability to delay entrance in M phase. We suggest also that other proteins known to be involved in checkpoint activation after hydroxyurea treatment are working in the pathway in which Dpb11 is involved
RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks.
Full text linkIn higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ?? onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Dpb11 and the 9-1-1 Complex Act Redundantly in Promoting Checkpoint Activation after Replication Stress
No full textFollowing DNA damage or replication stress, yeast cells phosphorylate and activate the Rad53 (hChk2) checkpoint protein, which is responsible for maintaining genome stability in these challenging conditions. The activation mechanism of the DNA damage checkpoint and of the replication checkpoint is different, but partially overlapping because most of checkpoint factors are shared between these pathways. For the activation of the two checkpoints the upstream kinase complex Mec1/Ddc2 (hATR/hATRIP) is required and both the PCNA-like complex and the replicative factor Dpb11 (hTopBP1) play a relevant role. We have previously shown that in UV damaged yeast cells, Dpb11 is required for the histone methylation-independent function of Rad9. Dpb11 has been also reported to be able to stimulate in vitro the Mec1 kinase activity. To better understand in vivo the function of Dpb11 in activating the apical kinase, we decided to study checkpoint activation after treatment with hydroxyurea (HU), an inhibitor of ribonucleotide reductase. In fact, HU induces Rad53 phosphorylation independently of the Rad9 adaptor protein allowing us to study the function of Dpb11 in the activation of Mec1 and its interplay with 9-1-1 complex (Ddc1-Mec3-Rad17). We show here that a ddc1Δdpb11-1 double mutant strain displays a Rad53 phosphorylation defect after HU treatment, similar to the one of a mec1-1 mutant. Moreover, the double mutant also lacks the phosphorylation of histone H2A. These observations suggest that Dpb11 and the PCNA-like complex act independently in promoting Mec1 activation. A similar phenotype can be observed in a dpb4Δddc1Δ strain carrying a deletion of a non-essential subunit of DNA polymerase ε, indicating that Dpb11 may be working together with Polε in this functio
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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