1,720,985 research outputs found
The role of PARPs and APLF in DNA interstrand crosslink repair
Full text linkOver the duration of every organismâs life its cells continuously face insults to their DNA. The accumulation of lesions results in cell death, inflammation and, following the activation of oncogenes or inactivating mutations of DNA repair factors, cancer formation. One particularly deleterious form of DNA damage are DNA interstrand crosslinks (ICLs), which prevent DNA strand seperation, therefore interfering with replication and transcription. Here we uncover a novel function for PARPs and the DNA repair protein APLF in the tolerance of human cells to DNA ICLs. Consistent with a role of PARPs in ICL repair, we observe ADP-ribosylation in response to the ICL inducing agent cis-platin. Furthermore, the inhibition of PARPs by Olaparib sensitizes human cells to ICLs. We identify the importance of PARP1 and PARP2 in these events and uncover that they are epistatic. We propose that PARP1 and PARP2 participate in ICL removal and that this is independent of the Fanconi Anemia pathway. The NHEJ repair factor APLF is recruited to chromatin in response to DNA ICLs and localizes to sites of laser induced damage in combination with the photreactive ICL inducing drug 4,5',8-trimethylpsoralen. Disruption of APLF sensitizes cells to ICLs. Additionally, the ADP-ribose binding tandem PBZ domain of APLF is essential for the restoration of MMC tolerance in aplfΔ cells. We suggest that APLF is an early responder to ICL formation. Together these data identify a novel role for PARP1, PARP2 and APLF in the tolerance of human cells to ICLs.</p
Study of DNA double strand break repair in Dictyostelium discoideum
Full text linkThe homologous recombination (HR) pathway contributes to genome integrity by mediating double strand break (DSB) repair using a homologous DNA sequence as a template. In mammals Rad51 and Brca2 are molecules central to this process. Little is known about HR repair in Dictyostelium. However, research previously conducted on DSB repair using this organism has shown that DSB repair pathways are highly conserved when compared to humans. This encouraged study of HR in this organism. In this study, through a bioinformatics search I have identified putative orthologues of most human HR proteins and most interestingly of BRCA2, which cannot be found in other lower eukaryotes used as models for DSB repair, such as the budding yeast S.cerevisiae. Brcp, the Dictyostelium BRCA2 ortholog, shows similar domain structure when compared to BRCA2-related proteins identified in other organisms. To verify the implication of HR proteins in DSB repair, I developed a method to monitor recruitment of DNA repair proteins on chromatin upon DSB induction. Findings of this study suggest that both Brcp and Rad51 get recruited to chromatin upon DSB induction and are therefore implicated in DSB repair in Dictyostelium. To further study Brcp function and based on findings suggesting that disruption of brcp might be lethal, I developed a novel system for specific and conditional depletion of endogenous Dictyostelium proteins. Utilizing this system, I conducted phenotypic studies in a strain depleted of Brcp to examine its role in DNA repair. Overall this study shows that the HR pathway in Dictyostelium shows great similarity to vertebrates, making Dictyostelium an appealing model for the study of DSB repair and specifically HR
Investigation and characterisation of poly(ADP-ribose)-binding proteins in Dictyostelium discoideum
Full text linkThe genome is under continual assault from endogenous and exogenous sources of DNA damage. The cell has therefore evolved a number of pathways to identify, signal, and ultimately repair DNA lesions. Poly(ADP-ribosyl)ation, the addition of multiple ADP-ribose moieties to proteins to form poly(ADP-ribose) (PAR) chains, has been implicated in several DNA repair pathways. One function of these PAR chains is to act as a scaffold for the recruitment of downstream repair proteins, suggesting the existence of protein domains that specifically bind PAR. Several of these domains have been identified, the best characterised being the PBZ and macro domains. We utilise in silico genome-wide searches to identify novel proteins containing PAR-binding domains in the simple eukaryotic model organism Dictyostelium discoideum. We identity three proteins with unannotated macro domains in Dictyostelium: DNA ligase III (Lig3), an aprataxin-like protein (APL), which also contains a PBZ domain, and Q54R54. The macro domain of APL was found to be circularly permuted compared to the other human and Dictyostelium macro domains; however, structure prediction by homology modelling indicated that it had retained the structure of a classical macro domain. We performed in vitro PAR-binding assays that indicated that the macro domains of both Lig3 and APL bind to PAR chains. Lig3 is enriched on DNA following the infliction of base damage, indicating its role in the SSBR pathway. However, the dependence of this pathway on PARylation has not been determined. In contrast, APL was found to be enriched on chromatin in response to DNA inter-strand cross-links and S-phase-associated double-strand breaks, which was reduced following mutation of both the PBZ and macro domains of APL. Furthermore, APL was found to be mono-ubiquitinated in response to DNA inter-strand cross-links, an event that is dependent on its macro domain region. These data indicate a role for APL in the repair of DNA inter-strand cross-links, or S-phase associated DNA damage, which would not be predicted from its homology to aprataxin, thereby suggesting a protein with novel characteristics in the DDR in Dictyostelium
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
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
The role of UHRF1 in Fanconi anemia pathway
Full text linkFanconi Anemia (FA) is a genetic disease caused by mutations in any one of the identified 16 genes. The corresponding proteins are known as FA proteins and compose the FA pathway, which is critical for the cellular response to DNA inter-strand cross-links (ICLs). Many efforts have been drawn to understand the individual role of the FA proteins, yet the mechanism of the initiation of the pathway remains elusive, and in particular, no sensor protein for ICLs has been identified. The aims of this study are to identify such a putative factor and to investigate the role of this factor in DNA ICL repair. We have analyzed the DNA binding activity of two up-stream players in the FA pathway, i.e. FANCD2 and FANCI, which do not seem to be able to detect the damage directly. We then designed a novel biochemical purification strategy using biotin-labeled ICL DNA to isolate ICL interacting proteins from HeLa nuclear extract. By using mass-spectrometry following the purification, we identified an E3 ligase named UHRF1. We cloned, expressed and purified UHRF1 from Sf9 cells. In vitro data demonstrate that UHRF1 specifically and directly interacts with cross-linked DNA. In vivo experiments indicate that UHRF1 participates in the FA pathway, potentially by recruiting FANCD2 to the sites of DNA damage, and the reduction of cellular levels of UHRF1 by RNA interference (RNAi) sensitizes cells to MMC. We also identified the SRA domain as the region of UHRF1 that is responsible for DNA ICL binding, and the interference of this region results in defects in the cells in ICL repair. With the unique feature of ICL DNA binding, UHRF1 is identified as a new player in the FA pathway. Future clinical studies will help to determine if UHRF1 is indeed an FA gene of which mutations can generate phenotypes of the disease. Also, a better understanding of the molecular mechanisms underlying UHRF1 in the FA pathway will enable us to develop better and more targeted modes of cancer therapies
Role of histones in DNA double-strand break repair in Dictyostelium
Full text linkCorrect repair of DNA double-strand breaks is crucial for maintenance of genome integrity. Despite data showing the importance of histones variants and histone post-translational modifications in the cellular response to DNA damage, there is still a lack of knowledge concerning the role of histone H3 and its variants as well as histone ADP-ribosylation in such processes. In this work Dictyostelium discoideum was employed as genetically tractable model organism to address the role of histone H3 variants and histone ADP-ribosylation in DNA double-strand break (DSB) repair. Vegetative cells lacking two out of three histone H3 variants – H3b and H3c, were shown not to be sensitive to DNA DSB. No evidence for altered DSB repair was found as phosphorylation of histone H2AX (a marker of DSB) and one of the pathways of DSB repair, non-homologous end joining, were not altered. Altogether, this work demonstrates that H3b and c variants are not required for overall DNA DSB repair in Dictyostelium. Among the core histones histone H2B was discovered to be the major acceptor of ADP-ribosylation by major ADP-ribosyl transferase involved in DSB repair, Adprt1a, in vitro. ADP-ribosylation in vitro was shown to occur on glutamate E18 with E19 being a potential regulator of this modification. Using an epitope-tagged overexpressed H2B, in vivo H2B ADP-ribosylation in response to DSBs was observed in Dictyostelium for the first time. Decreased ADP-ribosylation of epitope-tagged H2B mutated in both E18 and E19 residues was demonstrated. Overall, this work demonstrates the presence of the ADP-ribosylation of H2B in Dictyostelium in response to DSBs and identifies the major site of this modification
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
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