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Previous webinars > 7th Seminar, 03/07/20237th EDRA Webinar March 7, 2023 3.30 to 5 pm CET Zoom link
Pr Hilde Loge Nilsen - Head of research, Department of Microbiology, Oslo University Hospital
Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology DNA instability is a hallmark of aging and is linked to neurodegeneration, but the mechanisms are poorly understood. The integrity of both nuclear (n) and mitochondrial (mt) DNA is dependent on the DNA repair pathway Base Excision Repair (BER), which is the primary pathway for removing oxidative stress-induced DNA damage one of the most significant DNA repair processes in neurons. The BER pathway is initiated by DNA glycosylases, which recognize and excise damaged bases (e.g. oxidized) producing apurinic/apyrimidinic (AP) sites and single strand breaks (SSB). DNA glycosylase-mediated base excision is followed by a series of fine-tuned enzymatic steps that restore the DNA integrity by inserting undamaged bases. The purpose of this fine-tuning is to prevent the formation of toxic intermediates, such as SSBs. Unbalanced BER, where downstream enzymes do not match DNA glycosylase activity results in SSBs accumulation which can interfere with fundamental processes such as transcription and lead to cell death. I will present recent work where we have used C. elegans as a model organism, to show that unbalanced BER accompanies normal ageing and drives age-related neurodegeneration.
Short 15-min talks : Valentine Lagage - University of Oxford Adaptation delay causes a burst of mutation in bacteria responding to oxidative stress
Understanding the interplay between phenotypic plasticity and genetic adaptation is a long-standing focus of evolutionary biology. In bacteria, the oxidative stress response limits the formation of mutagenic reactive oxygen species (ROS) under diverse stress conditions. This suggests that the dynamics of the oxidative stress response are closely tied to the timing of the mutation supply that fuels genetic adaptation to stress. Here, we explored how mutation rates change in real-time in Escherichia coli cells during continuous hydrogen peroxide treatment in microfluidic channels. By visualising nascent DNA replication errors, we uncovered that sudden oxidative stress causes a burst of mutation. We developed a range of single-molecule and single-cell microscopy assays to determine how these mutation dynamics arise from phenotypic adaptation mechanisms. Signalling of peroxide stress by the transcription factor OxyR rapidly induces ROS scavenging enzymes. However, the adaptation delay leaves cells vulnerable to the mutagenic and toxic effects of hydroxyl radicals generated by the Fenton reaction. A spike in DNA repair activities counteracted accumulation of oxidative DNA damage during the adaptation delay. Prior stress exposure or constitutive OxyR induction allowed cells to avoid the burst of mutation. Similar observations for alkylation stress show that mutation bursts are a general phenomenon associated with adaptation delays.
Ali Nasrallah - Univ. Grenoble Alpes, INSERM, CEA, BIOMICS U1292, 38000 Grenoble, France
CRISPR-Cas9 mediated XPC disease modeling in human skin cells: RNAi- based biological screen to treat disease phenotype
The sun's DNA-damaging ultraviolet (UV) radiation remains the major extrinsic risk factor for skin cancer development. Nevertheless, mammalian cells exploit the presence of the nucleotide excision repair (NER) pathway as a protective shield to eliminate the accumulation of photoproducts generated by UVB radiation. Xeroderma Pigmentosum C (XPC) is a key multifunctional protein implicated in the NER pathway that acts as a sensor for helical distortions found in DNA lesions. Loss-of-function mutations in the XPC gene lead to various malignancies, including skin cancers. Photosensitivity and the buildup of DNA damage can both define the phenotype of XPC patients' cells. To date, there is no relevant and reproducible cellular model available that can mimic the course of the human XPC disease. We carried out, for the first time, XPC gene editing in several human skin cells (keratinocytes, fibroblasts, and melanocytes) using CRISPR-Cas9 technology. Full Characterization of XPC gene mutation was confirmed by western blot, quantitative PCR as well as sanger sequencing. The viability of XP-C cells post UV radiation was further quantified and the repair activity of DNA damage was assessed by Immunocytochemistry (ICC) via single-cell analysis. WT cells and XPC knockout cellular model were then used to map the specific proteomic signature of XPC cells at basal state and its modifications post UVB irradiation. For this, total proteomes of WT and XPC cells subjected or not to UVB irradiation (power, time) were prepared and analysed by mass spectrometry-based quantitative proteomics using isobaric labelling. Furthermore, we generated for the first time a full XPC KO reconstructed skin model to better mimic the natural skin niche. Interms of therapeutics, a kinome and phosphatome based biological screen was performed to revert/alleviate the disease phenotype based on inhibiting proteins that can mimic suppressor mutation phenomena. Novel hits will be further validated extensivelly on our 2D models and afterwards on our fully reconstructed XPC KO skin model.
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