Replication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue... Show moreReplication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue the deficits due to Rev1 loss remain unclear. Here, we report that loss of Rev1 leads to intense changes in metabolites and that this can be manipulated by NAD + supplementation. Autophagy decreases in Rev1-/- mouse embryonic fibroblasts (MEFs) and can be restored by supplementing the NAD+ precursor nicotinamide riboside (NR). The abnormal mitochondrial morphology in Rev1-/-MEFs can be partially reversed by NR supplementation, which also protects the mito-chondrial cristae from rotenone-induced degeneration. In nematodes rev-1 deficiency causes sensitivity to oxidative stress but this cannot be rescued by NR supplementation. In conclusion, Rev1 deficiency leads to metabolic dysregulation of especially lipid and nucleotide metabolism, impaired autophagy, and mitochondrial anomalies, and all of these phenotypes can be improved by NR replenishment in MEFs. Show less
All cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as... Show moreAll cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as transcription and replication and has the potential to drive mutagenesis. In turn, this may underlie inherited disorders and accelerate progression of diseases such as cancer and neurodegenerative disorders. The protection of cells and organisms against these devastating effects of DNA damage relies on the DNA damage response (DDR), which comprises a complex network of signaling and repair pathways that coordinate the sensing, signaling and repair of DNA lesions while accommodating suitable adjustments in for instance chromatin structure and cell cycle progression. Not only does the DDR dictate the appropriate repair pathway for several types of DNA damage, including DNA double-strand breaks (DSB), it also modulates replication fork surveillance mechanisms in response to DNA replication stress (RS). While many core proteins have been studied in detail, the full repertoire of factors involved in these pathways remains unknown. Clearly, extending our knowledge on regulators of the DDR will contribute to our understanding of the development, and possibly the treatment, of the numerous disorders that are associated with defects in the DDR. The research described in this thesis has successfully identified and characterized novel factors in DSB repair and the RS response. Show less
To advance our understanding of the histone acylation regulators and the response to DNA replication stress, we employ a variety of genetic screening techniques together with follow-up experiments... Show moreTo advance our understanding of the histone acylation regulators and the response to DNA replication stress, we employ a variety of genetic screening techniques together with follow-up experiments in yeast. By employing Epi-ID, we interrogated a common chromatin locus in the yeast deletion and DaMP collection. This was successful in identifying new histone acetylation and acylation regulators and we describe the crotonyltransferase activities of the ADA and NuA4 complexes. Increased crotonylation levels stimulated gene expression dependent on the activity of the HATs Gcn5 and Esa1, suggesting a role for these enzymes in the crotonylation-dependent transcriptional regulation. Additionally, extensive antibody validation studies revealed that non-acetyl pan-lysine specific acylation antibodies had a severe lack of specificity.To identify new regulators of replication stress, we developed a novel approach called Replication-Identifier (Repli-ID), which we used to investigate the accumulation of replicative polymerase ε near a barcoded origin of replication in mutant yeast collections. We demonstrate that this method is effective for studying DNA polymerase occupancy directly on chromatin and we identify new regulators of DNA replication fork progression/stability.Finally, we identify and characterize the Mediator complex in the replication stress response. We show how Mediator reduces replication stress by preventing R-loop formation. Show less
Jansen, J.G.; Tsaalbi-Shtylik, A.; Wind, N. de 2015
