Damages to the genetic materials arise throughout the lifespan of a cell, and elicit upregulation of DNA repair factors. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is part of a DNA repair protein complex that specialises in the repair of DNA base modifications and single-strand breaks (SSBs). TDP1 removes a broad spectrum of chemical adducts from the 3' end of a DNA strand break, including topoisomerase 1 (TOP1) peptide, during DNA transcription and replication. Inactivation or deletion of TDP1 is associated with cerebellar dysfunction and degeneration, with remarkably little extraneurological manifestation. The reason for the selective dependence of the cerebellar neurons on TDP1 activity is not clear. It was hypothesised that the TDP1 activity is upregulated in tissues with high levels of SSBs, either from DNA transcriptional activity, or reactive oxygen species (ROS)-induced damage. The aim of this doctoral project was therefore to identify and characterise the cellular mechanisms that regulate TDP1 activity. Our lab has previously shown that the Nterminus domain (NTD) of TDP1 covalently interacts with DNA ligase 3α. In this thesis, evidence has been presented to show that this interaction is regulated by the putative ATM/ATR/DNA-PK phosphorylation site, serine 81, to prolong TDP1 half-life, and enhance cellular survival after genotoxic stress. A second post-translational modification in the NTD by SUMOylation of the K111 residue was identified, enlightening a mechanism by which TDP1 is recruited to sites of transcription-mediated SSBs. To investigate the requirement for TDP1 in cells under high levels of oxidative stress, I have developed a mouse cellular model whereby the levels of endogenous ROS can be modulated by overexpression of the human anti-oxidant enzyme superoxide dismutase 1 (SOD1) or its toxic mutant SOD1G93A. Overexpression of SOD1G93A in Tdp1-/- MEFs induces accumulation of chromosomal SSBs and decreases survival after H2O2 challenge, while overexpression of SOD1 has a protective effect. Besides repair of ROS-induced TOP1-cc in the nucleus, TDP1 also repairs mitochondrial topoisomerase 1-mediated DNA breaks. This role is required during transcription and assembly of mitochondrial subunits of the electron transfer chain complexes, and has direct impact on mitochondrial respiration and ROS production. Collectively, these data provide mechanistic insights into regulation of TDP1-mediated chromosomal and mitochondrial DNA repair.