Regulation of replication-linked functions by PCNA and SUMO

Genomic integrity largely depends on the accurate replication and faithful transmission of the genetic information to the progeny each time a cell divides. To ensure the fidelity of these fundamental processes, highly sophisticated protein networks have evolved. This study investigated how the diverse mechanisms for maintaining genomic integrity are integrated and coordinated at the replication fork. In the first part, the roles of post-translational modifications with ubiquitin and SUMO in regulating replication through DNA lesions were investigated. Previous work in S. cerevisiae showed that post-translational modifications of the replication factor PCNA control DNA repair activities of the replisome. PCNA is a homotrimeric ring-shaped protein that encircles DNA and confers processivity to DNA polymerases during DNA synthesis. Moreover, being devoid of enzymatic activity, PCNA is perfectly suited to act as a platform for recruitment of factors to the replication fork, including DNA repair enzymes, chromatin remodelers and replication regulators. PCNA modifications by ubiquitin govern two distinct modes of lesion bypass, either channeling the repair processes into error-prone translesion synthesis by recruiting specialized polymerases, or promoting an error-free mechanism involving a template switch to the sister chromatid. In addition, PCNA-modification by SUMO inhibits the third major bypass mechanism, namely recombinational repair, by recruiting the anti-recombinogenic helicase Srs2. In this study, the importance and universality of PCNA-modifications could be demonstrated by showing that PCNA ubiquitylation in human and chicken cells is well conserved. Interestingly, SUMO modification appeared less preserved, indicating that the pathways controlled by PCNA modifications are used to different degrees throughout species. A further finding of this thesis was the identification of DNA polymeras

PaaSword: A Data Privacy and Context-aware Security Framework for Developing Secure Cloud Applications - Technical and Scientific Contributions

Most industries worldwide have entered a period of reaping the benefits and opportunities cloud offers. At the same time, many efforts are made to address engineering challenges for the secure development of cloud systems and software.With the majority of software engineering projects today relying on the cloud, the task to structure end-to-end secure-by-design cloud systems becomes challenging but at the same time mandatory. The PaaSword project has been commissioned to address security and data privacy in a holistic way by proposing a context-aware security-by-design framework to support software developers in constructing secure applications for the cloud. This chapter presents an overview of the PaaSword project results, including the scientific achievements as well as the description of the technical solution. The benefits offered by the framework are validated through two pilot implementations and conclusions are drawn based on the future research challenges which are discussed in a research agenda

Identifying Exoplanets with Deep Learning. V. Improved Light Curve Classification for TESS Full Frame Image Observations

The TESS mission produces a large amount of time series data, only a small fraction of which contain detectable exoplanetary transit signals. Deep learning techniques such as neural networks have proved effective at differentiating promising astrophysical eclipsing candidates from other phenomena such as stellar variability and systematic instrumental effects in an efficient, unbiased and sustainable manner. This paper presents a high quality dataset containing light curves from the Primary Mission and 1st Extended Mission full frame images and periodic signals detected via Box Least Squares (Kov\'acs et al. 2002; Hartman 2012). The dataset was curated using a thorough manual review process then used to train a neural network called Astronet-Triage-v2. On our test set, for transiting/eclipsing events we achieve a 99.6% recall (true positives over all data with positive labels) at a precision of 75.7% (true positives over all predicted positives). Since 90% of our training data is from the Primary Mission, we also test our ability to generalize on held-out 1st Extended Mission data. Here, we find an area under the precision-recall curve of 0.965, a 4% improvement over Astronet-Triage (Yu et al. 2019). On the TESS Object of Interest (TOI) Catalog through April 2022, a shortlist of planets and planet candidates, Astronet-Triage-v2 is able to recover 3577 out of 4140 TOIs, while Astronet-Triage only recovers 3349 targets at an equal level of precision. In other words, upgrading to Astronet-Triage-v2 helps save at least 200 planet candidates from being lost. The new model is currently used for planet candidate triage in the Quick-Look Pipeline (Huang et al. 2020a,b; Kunimoto et al. 2021).Comment: accepted for publication in AJ. code can be found at: https://github.com/mdanatg/Astronet-Triage and data can be found at: https://zenodo.org/record/741157

FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ\u27s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage

Obtaining and Characterizing Alginate/k-Carrageenan Hydrogel Cross-Linked with Adipic Dihydrazide

The aim of this paper is obtaining and characterizing hydrogels based on different ratios of oxidized alginate (oA) and k-carrageenan (C), chemically cross-linked with adipic dihydrazide (adh). The alginate (A) was first oxidized with sodium metaperiodate in order to transform it into the dialdehyde derivative, a more reactive compound than alginate. A known procedure for oxidation of alginate with sodium metaperiodate in ethanol-water in order to improve alginate reactivity by transforming the hydroxyl end-groups into dialdehyde was used, preceded by a partially cleavage of the alginate chains. In the second stage, the mixture of dialdehydic derivative of oxidized alginate, k-carrageenan and glycerol subjected to reaction with adipic dihydrazide leads to a Semi-Interpenetrated Network covalently cross-linked alginate/k-carrageenan hydrogel (oACadh), based on the dihydrazone compound which is responsible for the chemical cross-linking. Pure alginate, k-carrageenan, oxidized alginate, adipic dihydrazide and the cross-linked hydrogel were characterized by: FTIR, XRD, and SEM

Heterozygous De Novo UBTF Gain-of-Function Variant Is Associated with Neurodegeneration in Childhood.

Ribosomal RNA (rRNA) is transcribed from rDNA by RNA polymerase I (Pol I) to produce the 45S precursor of the 28S, 5.8S, and 18S rRNA components of the ribosome. Two transcription factors have been defined for Pol I in mammals, the selectivity factor SL1, and the upstream binding transcription factor (UBF), which interacts with the upstream control element to facilitate the assembly of the transcription initiation complex including SL1 and Pol I. In seven unrelated affected individuals, all suffering from developmental regression starting at 2.5-7 years, we identified a heterozygous variant, c.628G\u3eA in UBTF, encoding p.Glu210Lys in UBF, which occurred de novo in all cases. While the levels of UBF, Ser388 phosphorylated UBF, and other Pol I-related components (POLR1E, TAF1A, and TAF1C) remained unchanged in cells of an affected individual, the variant conferred gain of function to UBF, manifesting by markedly increased UBF binding to the rDNA promoter and to the 5\u27- external transcribed spacer. This was associated with significantly increased 18S expression, and enlarged nucleoli which were reduced in number per cell. The data link neurodegeneration in childhood with altered rDNA chromatin status and rRNA metabolism

Peroxynitrate formed during a transient episode of brain ischemia increases endothelium-derived hyperpolarization-type dilations in thromboxane/prostaglandin receptor stimulated rat cerebral arteries

Aim Increased thromboxane A2 and peroxynitrite are hallmarks of cerebral ischemia/reperfusion (I/R). Stimulation of thromboxane/prostaglandin receptors (TP) attenuates endothelium-derived hyperpolarization (EDH). We investigated whether EDH-type middle cerebral artery (MCA) relaxations following TP stimulation are altered after I/R and the influence of peroxynitrite. Methods Vascular function was determined by wire myography after TP stimulation with the thromboxane A2 mimetic 9,11-Dideoxy-9α,11α-methano-epoxy prostaglandin F2α (U46619) in MCA of Sprague-Dawley rats subjected to MCA occlusion (90 min)/reperfusion (24 h) or sham operation, and in non-operated (control) rats. Some rats were treated with saline or the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) (20 mg kg-1). Protein expression was evaluated in MCA and in human microvascular endothelial cells submitted to hypoxia (overnight)/reoxygenation (24 h) (H/R) using immunofluorescence and immunoblotting. Results In U46619-preconstricted MCA, EDH-type relaxation by the proteinase-activated receptor 2 agonist serine–leucine–isoleucine–glycine–arginine–leucine–NH2 (SLIGRL) was greater in I/R than sham rats due to an increased contribution of small-conductance calcium-activated potassium channels (SKCa), which was confirmed by the enlarged relaxation to the SKCa activator N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine. I/R and H/R induced endothelial protein tyrosine nitration and filamentous-actin disruption. In control MCA, either cytochalasin D or peroxynitrite disrupted endothelial filamentous-actin and augmented EDH-type relaxation. Furthermore, peroxynitrite decomposition during I/R prevented the increase in EDH-type responses. Conclusion Following TP stimulation in MCA, EDH-type relaxation to SLIGRL is greater after I/R due to endothelial filamentous-actin disruption by peroxynitrite, which prevents TP-induced block of SKCa input to EDH. These results reveal a novel mechanism whereby peroxynitrite could promote postischemic brain injury

A Role for PCNA Ubiquitination in Immunoglobulin Hypermutation

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNA(K164R) mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases

The prognostic value of p53 mutation in pediatric marrow hypoplasia

<p>Abstract</p> <p>Background</p> <p>The tumor suppressor gene p53 is involved in the control of cell proliferation, particularly in stressed cells. p 53 gene mutations are the most frequent genetic event found in human cancers. Fanconi Anemia (FA) is the most common representative of inherited bone marrow failure syndromes (IBMFS) with a leukemic propensity. P 53 DNA alteration has not been studied before in Egyptian children with FA.</p> <p>Patients and methods</p> <p>we investigated p53 mutation in the bone marrow and peripheral blood of forty children, FA (n = 10), acquired aplastic anemia (AAA) (n = 10), and immune thrombocytopenia (ITP) as a control (n = 20), using real-time PCR by TaqMan probe assay</p> <p>Results</p> <p>Mutation of p53 gene was demonstrated in the BM of 90% (9/10) of children with FA, compared to 10% (1/10) in AAA (p < 0.001), while, no p53 DNA mutation was seen in the control group. A positive correlation between DNA breakage and presence of p53 mutation was seen in FA (p < 0.02, r0.81).</p> <p>Conclusion</p> <p>mutation of p53 gene in hypoplastic marrow especially FA may represent an early indicator of significant DNA genetic alteration with cancer propensity.</p