Mechanistic insight into oxidative stress-mediated genome instability Summary: Genome instability is characterized by genetic alterations ranging from DNA base mutations to chromosome rearrangements that are drivers of many inherited human diseases, various types of cancer and premature ageing. Chromosome instability, especially, results from inaccurate chromosomal segregation caused by telomere and centromere dysfunction. Numerous epidemiologic studies have highlighted the central role of oxidative stress exposures in the occurrence of telomere and centromere dysfunction. Critically, however, the mechanisms underlying the dysfunction are not clearly understood. Oxidative stress results from an imbalance between the production of reactive oxygen species and cellular antioxidant defenses. It arises from endogenous sources as well as from environmental sources (mitochondria metabolism, UV light, air pollution, cigarette smoke). Its ubiquity highlights the importance of properly understanding its impacts on human health. A major impact of oxidative stress is the induction of oxidative DNA damage that are repaired by the base excision repair (BER) pathway in which poly(ADP-ribose) polymerases (PARPs) are major actors. PARP1 and PARP2 are responsible for the poly(ADP-ribosyl)ation, a post-translational modification of proteins that modulates the recruitment and interactions of their protein targets. The goals of this proposal are to (i) uncover the mechanisms of oxidative stress-mediated genome instability with a focus on its impact on telomeres and centromeres, two genomic loci crucial for genome stability and (ii) decipher the contribution of PARP enzymes in the protection of telomeric and centromeric DNA upon oxidative DNA damage. More specifically, we aim to evaluate the impact of chronic induction of oxidized DNA bases and BER single strand break intermediates on centromere and telomere integrity. We will also identify and assess the impact of poly(ADP-ribosyl)ation on centromeric and telomeric protein targets. To this end, we will leverage a unique and innovative chemoptogenetic tool that induces oxidative DNA damage locally at telomeres and at centromeres without impacting the rest of the genome. This will allow us to unequivocally link phenotypic changes and PARP dependent mechanisms to the telomeric or centromeric lesions. These projects will fill a long-standing gap of knowledge on how poly(ADP-ribosyl)ation orchestrates DNA repair at two crucial regions of the genome. They will also shed light on how oxidative stress, a ubiquitous factor of genome instability, can drive numerous human diseases. Ultimately, our work will contribute to the development of novel therapeutic strategies targeting specific regions of the genome and inform the rational design and use of the PARP inhibitors already widely used in cancer treatments.