Investigation of DNA repair at gene regulatory regions

The nuclear mitotic apparatus protein (NuMA) is classically characterised for its role in mitotic spindle organisation, but its functions in genome maintenance outside mitosis remain incompletely understood. This thesis identifies NuMA as a regulator of genome integrity, influencing oxidative DNA damage at regulatory chromatin, and the cellular response to topoisomerase 1(TOP1)-induced DNA-protein crosslinks.
To investigate NuMA function in a post-mitotic context, an mESC-derived neuronal cell line was generated in which endogenous NuMA was tagged with FKBP12F36V-FLAG, enabling acute depletion upon dTAG-13 treatment. This system was used to test NuMA’s protective role against DNA damage arising during active cytosine demethylation. Genome-wide mapping of abasic (AP) sites revealed selective accumulation at gene regulatory regions, including promoters and enhancers. Genes showing the strongest damage increases at regulatory regions were enriched for neuronal and synapse-related functions, linking NuMA-dependent chromatin protection to pathways relevant for neuronal physiology. Furthermore, NuMA localised to the same regulatory regions, placing it near vulnerable chromatin.
Building on the observation that AP lesions and strand breaks can stabilise TOP1 cleavage complexes (TOP1ccs), NuMA’s role in TOP1cc resolution was next examined in both proliferating and post-mitotic cells. NuMA depletion increased basal and camptothecin-induced TOP1cc accumulation and elevated DNA strand breaks. Unexpectedly, despite higher lesion burden, NuMA-deficient cells showed attenuated DNA damage signalling with reduced checkpoint activation and impaired p53-dependent apoptosis, resulting in apparent resistance to TOP1 poisoning. Biochemical analyses further demonstrated persistent PARylation of TOP1ccs in the absence of NuMA, consistent with defective lesion processing and impaired generation of signalling-competent repair intermediates.
These results support a role for NuMA in maintaining genome integrity across multiple lesion types, by protecting vulnerable regulatory chromatin and promoting appropriate processing and signalling of TOP1-induced DNA damage.